Compositions and methods for intravitreal delivery of polynucleotides to retinal cones

ABSTRACT

Methods and compositions are provided for intravitreally delivering a polynucleotide to cone photoreceptors. Aspects of the methods include injecting a recombinant adeno-associated virus comprising a polynucleotide of interest into the vitreous of the eye. These methods and compositions find particular use in treating ocular disorders associated with cone dysfunction and/or death.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of U.S. patentapplication Ser. No. 15/554,664, filed Aug. 30, 2017, which is a U.S.National Phase Application of International Patent Application No.PCT/US2016/020482, filed Mar. 2, 2016; which claims the benefit under 35U.S.C. § 119(e) of U.S. Provisional Application No. 62/127,194, filed onMar. 2, 2015, and U.S. Provisional Application No. 62/134,466, filed onMar. 17, 2015; each of which is incorporated by reference herein in itsentirety.

STATEMENT REGARDING SEQUENCE LISTING

The Sequence Listing associated with this application is provided intext format in lieu of a paper copy, and is hereby incorporated byreference into the specification. The name of the text file containingthe Sequence Listing is AVBI_006_04 US_ST25.txt. The text file is 74 KB,was created on May 5, 2021, and is being submitted electronically viaEFS-Web.

FIELD OF THE INVENTION

This invention pertains to viral-based gene therapy of retinaldisorders.

BACKGROUND OF THE INVENTION

Photoreceptors are a specialized type of neuron found in the retina thatare capable detecting light and converting that light signal intoelectrical signals. There are two types of photoreceptors in the retina:rod photoreceptors, which are more sensitive to light and hence supportvision in dim lighting; and cone photoreceptors, which are sensitive tospecific wavelengths of light and hence support the perception of color,and which respond faster to stimuli than rods so perceive finer detailand more rapid changes in images than rods and hence support high acuityvision.

A number of vision disorders are associated with a loss of viability orfunction of the cone photoreceptors, including, for example, thoseassociated with defects within cones, i.e. cone-intrinsic defects, suchas Stargardt's macular dystrophy, cone dystrophy, cone-rod dystrophy,Spinocerebellar ataxia type 7, and Bardet-Biedl syndrome-1, as well ascolor vision disorders, including achromotopsia, blue cone monochromacy,and protan, deutan, and tritan defects; and those that are associatedwith retinal disorders that affect the central macula, such asage-related macular degeneration, macular telangiectasia, retinitispigmentosa, diabetic retinopathy, retinal vein occlusions, glaucoma,Sorsby's fundus dystrophy, adult vitelliform macular dystrophy, Best'sdisease, and X-linked retinoschisis. It is expected that these cone celldisorders may be treated by delivering to cone photoreceptors atherapeutic gene that, when expressed by the cone photoreceptors,complements the deficiency and “rescues” the cone cell viability and/orfunction.

The highest density of cone photoreceptors exist at the 1.5 mmdepression located in the center of the macula of the retina. Thisregion, called the “fovea centralis” or “foveal pit”, is responsible forsharp central vision (also called foveal vision), which is necessary inhumans for activities where visual detail is of primary importance, suchas reading and driving. The fovea centralis consists of two sub-regions:the foveola, a 0.35 mm diameter rod-free region of retina at the centerof the pit; and the fovea, a 1.5 mm-diameter cone-enriched region ofretina that surrounds the foveola and forms the slopes of the pit.Surrounding the fovea centralis is the parafovea, which forms the lip ofthe depression and is comprised of all cells of the retina, conephotoreceptors being represented in reduced numbers relative to in thefovea centralis. Beyond the parafovea is the perifovea, a region ofretina which contains an even more diminished density of cones. Becausecone cells of the fovea constitute the vast majority of conephotoreceptors in the retina, these cells are ideal target recipients oftherapeutic genes delivered for the treatment of cone-associateddisorders (Oster 1935).

Some success at delivering genes to cells of the retina has beenachieved by employing viral vectors such as adeno-associated virus (AAV)or lentivirus. However, these vectors must be administered by subretinalinjection, a procedure that disrupts the structure of the retina andcarries with it a risk of creating additional damage to retinal tissuethat is often already damaged by the disorder being treated. Onealternative is to deliver the viral vector to the retina intravitreally,i.e., by injecting the vector into the vitreous of the eye and hopingthat the vector permeates the retina and transduces the retinal cells.However, as demonstrated by the art, foveal cone cells are notoriouslyresistant to transduction by viral vectors delivered intravitreally tothe retina.

Thus, there is a need in the art for viral vectors that transduce conecells with high efficiency when delivered from the vitreous of the eye.The present invention addresses these issues.

SUMMARY OF THE INVENTION

Methods and compositions are provided for intravitreally delivering apolynucleotide to cone photoreceptors. Aspects of the methods includeinjecting a recombinant adeno-associated virus comprising apolynucleotide of interest into the vitreous of the eye. These methodsand compositions find particular use in treating ocular disordersassociated with cone dysfunction and/or death.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention is best understood from the following detailed descriptionwhen read in conjunction with the accompanying drawings. It isemphasized that, according to common practice, the various features ofthe drawings are not to-scale. On the contrary, the dimensions of thevarious features are arbitrarily expanded or reduced for clarity.Included in the drawings are the following figures.

FIG. 1 illustrates how intravitreally-delivered AAV2 variant AAV2-7m8transduces retinal cells in the fovea centralis and parafovea ofprimates more efficiently than intravitreally-delivered AAV2. 5×10¹¹vector genomes of AAV2.CMV.GFP (upper left); AAV-2.5T.CMV.GFP (upperright) (Excoffon K. J., et al. 2009. Proc. Natl. Acad. Sci. U.S.A106:3865-3870); (lower left) AAV2-7.8.CMV.GFP (Dalkara D, et al. SciTransl Med. 2013 Jun. 12; 5(189):189ra76); or AAV-ShH10.CMV.GFP (lowerright) (Klimczak R R et al. PLoS One. 2009 Oct. 14; 4(10):e7467) wasinjected into the vitreous of an African green monkey in a volume of 50uL, and GFP expression was observed 8 weeks later by OCT fluorescenceimaging in vivo.

FIG. 2 illustrates how the robustly the AAV2-7m8 capsid transducesfoveal cones of primates. (a-b) AAV2-7m8.MNTC.GFP was injected into thecentral vitreous of a baboon and expression was observed (a) 5 weeks and(b) 8 weeks later by fundus fluorescence. (c and d) Natural GFPfluorescence within a 15 micron section of the fovea at approximately 6months after injection with AAV2-7m8.MNTC.GFP at low magnification (c)and high magnification (d).

FIG. 3 illustrates robust and cone-specific gene expression in the conesof a mouse retina following intravitreal injection of AAV-7m8 deliveredMNTC.GFP. (a-b) Examples of GFP fluorescence 11 weeks after micereceived intravitreal injections of 5.04×10¹⁰ vector genomes viaintravitreal injection. (c-e) retinas were harvested for histology 14weeks after injection and cone outer segments were labeled with anantibody to L/M opsin (red). In (c) the red channel is turned off soonly the native GFP is visible, (d) is the same image with the redchannel on to allow visualization of cone outer segments. Comparison of(c) and (d) shows that most if not all cones were transduced by thevirus. (e) Image from the same retina as in c and d from different angleshowing profiles of cone photoreceptors.

FIG. 4A-4B illustrates gene expression directed by the pMNTC regulatorycassette in the cones of the Mongolian gerbil retina. 1×10¹⁰-2×10¹⁰vector genomes of virus carrying GFP under the control of the CMV,pR2.1, or MNTC promoter were injected in a volume of 5 uL into thevitreous of a Mongolian gerbil, and GFP expression visualized at thedesignated time points by fundus fluorescence imaging. (a) Expression ofGFP directed by AAV2-7m8.CMV.GFP and AAV2-7m8.MNTC.GFP, visualized 4weeks after intravitreal administration. Gerbils 12-10, 12-11, and 12-12were injected with AAV2-7m8.CMV.GFP, while gerbils 12-13, 12-14, and12-15 were injected with AAV2-7m8.MNTC.GFP. OD, oculus dexter (righteye). OS, oculus sinister (left eye). (b) Expression of GFP directed byAAV2-7m8.pR2.1.GFP and AAV2-7m8.MNTC.GFP, 4 and 8 weeks later asdetected by fundus fluorescence imaging.

FIG. 5A-5D demonstrate that the pMNTC regulatory cassette provides formore robust gene expression in foveal cones of primates than the conepromoter pR2.1. 5×10¹¹ vector genomes of AAV2-7m8.MNTC.GFP orAAV2-7m8.pR2.1.GFP were injected in a volume of 50 uL into the vitreousof African Green Monkeys as indicated (AAV2-7m8.MNTC.GFP into animals271 and 472; AAV2-7m8.pR2.1.GFP into animals 500 and 509). Retinas werevisualized in vivo at (a) 2 weeks, (b) 4 weeks, (c) 8 weeks, and (d) 12weeks for GFP using a fundus fluorescence camera (a, b, c, d) orautofluorescence on Heidelberg Spectralis OCT (a, b; data not shown forweeks 8 and 12). OD, oculus dexter (right eye). OS, oculus sinister(left eye).

FIG. 6A-6D demonstrate the contribution of each of the optimized pMNTCelements to the more robust expression observed. (a) The pMNTC and pR2.1expression cassettes. (b) The experimental expression cassettes, inwhich each element in pMNTC is replaced one-by-one by the correspondingelement in pR2.1. (c,d) Expression of the luciferase transgene in theretinas of gerbils intravitreally injected with each of the testarticles (n=6-8 eyes per construct) as detected (c) 4 weeks and (d) 8weeks after injection by IVIS imaging. “7m8.CMV” served as the positivecontrol.

FIG. 7 illustrates cone-specific gene expression directed by the pR2.1regulatory cassette in a non-human primate (NHP). 5×10¹¹ vector genomesof AAV2-7m8.pR2.1.GFP were injected in a volume of 50 uL into thevitreous of an African Green Monkey. GFP transgene expression wasobserved by stereo fluorescence microscopy of an 8 μm cross-section ofthe retina. GFP was stained with an anti-GFP antibody (green; chickenpolyclonal; Abcam Cat #13970); opsin cone cells were stained with ananti-L/M Opsin antibody specific for opsin cones (red; rabbitpolyclonal; Abcam Cat #5405); rod cells were stained with ananti-rhodopsin antibody (1D4 pink; mouse monoclonal; Abcam Cat #5417);and nuclei were stained with Dapi (blue/all nuclei; Invitrogen REF#D21490. GFP staining co-localized with L/M opsin staining but notrhodopsin staining. GFP transgene expression was present in L/M-opsincones across the photoreceptor layer, but GFP transgene expression wasnot observed in rods. Arrows indicate illustrative cone cellsdouble-stained for both GFP and opsin.

DETAILED DESCRIPTION OF THE INVENTION

Methods and compositions are provided for intravitreally delivering apolynucleotide to cone photoreceptors. Aspects of the methods includeinjecting a recombinant adeno-associated virus comprising thepolynucleotide of interest into the vitreous of the eye. These methodsand compositions find particular use in treating ocular disordersassociated with cone dysfunction and/or death. These and other objects,advantages, and features of the invention will become apparent to thosepersons skilled in the art upon reading the details of the compositionsand methods as more fully described below.

Before the present methods and compositions are described, it is to beunderstood that this invention is not limited to particular method orcomposition described, as such may, of course, vary. It is also to beunderstood that the terminology used herein is for the purpose ofdescribing particular embodiments only, and is not intended to belimiting, since the scope of the present invention will be limited onlyby the appended claims.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimits of that range is also specifically disclosed. Each smaller rangebetween any stated value or intervening value in a stated range and anyother stated or intervening value in that stated range is encompassedwithin the invention. The upper and lower limits of these smaller rangesmay independently be included or excluded in the range, and each rangewhere either, neither or both limits are included in the smaller rangesis also encompassed within the invention, subject to any specificallyexcluded limit in the stated range. Where the stated range includes oneor both of the limits, ranges excluding either or both of those includedlimits are also included in the invention.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, some potential andpreferred methods and materials are now described.

All publications mentioned herein are incorporated herein by referenceto disclose and describe the methods and/or materials in connection withwhich the publications are cited. It is understood that the presentdisclosure supersedes any disclosure of an incorporated publication tothe extent there is a contradiction.

As will be apparent to those of skill in the art upon reading thisdisclosure, each of the individual embodiments described and illustratedherein has discrete components and features which may be readilyseparated from or combined with the features of any of the other severalembodiments without departing from the scope or spirit of the presentinvention. Any recited method can be carried out in the order of eventsrecited or in any other order which is logically possible.

It is further noted that the claims may be drafted to exclude anyoptional element. As such, this statement is intended to serve asantecedent basis for use of such exclusive terminology as “solely”,“only” and the like in connection with the recitation of claim elements,or the use of a “negative” limitation.

It must be noted that as used herein and in the appended claims, thesingular forms “a”, “an”, and “the” include plural referents unless thecontext clearly dictates otherwise. Thus, for example, reference to “acell” includes a plurality of such cells and reference to “thepolynucleotide” includes reference to one or more polynucleotides andequivalents thereof, e.g. nucleic acid sequences, known to those skilledin the art, and so forth.

The publications discussed herein are provided solely for theirdisclosure prior to the filing date of the present application. Nothingherein is to be construed as an admission that the present invention isnot entitled to antedate such publication by virtue of prior invention.Further, the dates of publication provided may be different from theactual publication dates which may need to be independently confirmed.

Definitions

A “vector” as used herein refers to a macromolecule or association ofmacromolecules that comprises or associates with a polynucleotide andwhich can be used to mediate delivery of the polynucleotide to a cell.Illustrative vectors include, for example, plasmids, viral vectors(virus or the viral genome thereof), liposomes, and other gene deliveryvehicles.

By a “virus” it is meant a viral particle comprising a viral capsid anda viral genome. For example, an adeno-associated virus refers to a viralparticle comprising at least one adeno-associated virus capsid proteinor variant thereof and an encapsidated adeno-associated virus vectorgenome or variant thereof.

By a viral “capsid” it is meant the protein shell of a virus. Viralcapsids typically comprise several oligomeric structural subunits madeof protein called protomers. The capsid encloses, or “encapsidates”, thegenetic material, or “genome”, of the virus. In some viruses, the capsidis enveloped, meaning that the capsid is coated with a lipid membraneknown as a viral envelope.

By a viral “genome” (referred to interchangeably herein as “viralgenome”, “viral vector DNA” and “viral DNA”), it is meant apolynucleotide sequence comprising at least one, and generally two,viral terminal repeats (e.g. inverted terminal repeats (ITRs), longterminal repeats (LTR)) at its ends.

By a “recombinant viral genome” it is meant a viral genome comprising aheterologous nucleic acid sequence and at least one, and generally two,viral terminal repeats at its ends. By a “recombinant virus” it is meanta viral particle comprising a recombinant viral genome.

As used herein, the term “heterologous” means derived from agenotypically distinct entity from that of the rest of the entity towhich it is being compared. For example, a polynucleotide introduced bygenetic engineering techniques into a plasmid or vector derived from adifferent species, e.g. a viral genome, is a heterologouspolynucleotide. As another example, a promoter removed from its nativecoding sequence and operatively linked to a coding sequence with whichit is not naturally found linked is a heterologous promoter. As a thirdexample, a heterologous gene product, e.g. RNA, protein, is a geneproduct not normally encoded by a cell in which it is being expressed.

The term “replication defective” as used herein relative to the virusesof the disclosure refers to a virus that cannot independently replicateand package its genome. For example, when a cell of a subject isinfected with recombinant virions, the heterologous gene is expressed inthe infected cells; however, due to the fact that the infected cellslack AAV rep and cap genes and accessory function genes, the recombinantvirus is not able to replicate further.

The term “AAV” is an abbreviation for adeno-associated virus. When usedherein, the term AAV may be used to refer to the virus itself orderivatives thereof, e.g. the viral capsid, the viral genome, and thelike. The term “AAV” encompasses all subtypes, both naturally occurringand recombinant forms, and variants thereof except where requiredotherwise.

By “naturally occurring” or “wild-type” AAV it is meant anyadeno-associated virus or derivative thereof comprising a viral capsidthat consists of viral capsid proteins that occur in nature.Non-limiting examples of naturally occurring AAV include AAV type 1(AAV-1), AAV type 2 (AAV-2), AAV type 3 (AAV-3), AAV type 4 (AAV-4), AAVtype 5 (AAV-5), AAV type 6 (AAV-6), AAV type 7 (AAV-7), AAV type 8(AAV-8), AAV9, AAV10, AAV11, AAV12, rh10, avian AAV, bovine AAV, canineAAV, equine AAV, primate AAV, non-primate AAV, and ovine AAV. “PrimateAAV” refers to AAV that infect primates, “non-primate AAV” refers to AAVthat infect non-primate mammals, “bovine AAV” refers to AAV that infectbovine mammals, etc.

By an “AAV variant” or a “variant AAV” it is meant to include an AAVviral particle comprising a variant, or mutant, AAV capsid protein.Examples of variant AAV capsid proteins include AAV capsid proteinscomprising at least one amino acid difference (e.g., amino acidsubstitution, amino acid insertion, amino acid deletion) relative to acorresponding parental AAV capsid protein, i.e. an AAV capsid proteinfrom which it was derived, a wild type AAV capsid protein, etc., wherethe variant AAV capsid protein does not consist of an amino acidsequence present in a naturally occurring AAV capsid protein. Inaddition to differing structurally, i.e. at the sequence level, from thecorresponding parental AAV, the AAV variant may differ functionally fromthe corresponding parental AAV. Put another way, the variant capsidprotein comprising the at least one amino acid difference relative to acorresponding parental AAV capsid protein may confer functionalcharacteristics on the AAV variant that are not possessed by thecorresponding parental AAV. For example, the AAV variant may have adifferent cellular tropism, i.e. a different affinity for and/or abilityto infect a particular type of cell, e.g. the AAV variant may bind to acell, e.g. a retinal cell, with an increased (or decreased) affinitythan the parental AAV, and/or infect/transduce a cell, e.g. a retinalcell, with an increased (or decreased) efficiency than the parental AAVsuch that more (or less) cells of a cell population istransduced/infected with the same titer of viral particles. As a secondexample, the AAV variant may have a greater (or lesser) affinity forantibodies produced by the host animal, e.g. the AAV variant may bindwith greater (or lesser) affinity to neutralizing antibodies and becleared from the host tissue to a greater (or lesser) extent.

By “recombinant AAV”, or “rAAV” it is meant to include any AAV thatcomprises a heterologous polynucleotide sequence in its viral genome. Ingeneral, the heterologous polynucleotide is flanked by at least one, andgenerally by two naturally occurring or variant AAV inverted terminalrepeat sequences (ITRs). The term rAAV vector encompasses both rAAVvector particles and rAAV vector plasmids. Thus, for example, an rAAVthat comprises a heterologous polynucleotide sequence would be an rAAVthat includes a nucleic acid sequence not normally included in anaturally-occurring, wild-type AAV, for example, a transgene (e.g. anon-AAV RNA-coding polynucleotide sequence, non-AAV protein-codingpolynucleotide sequence), a non-AAV promoter sequence, a non-AAVpoly-adenylation sequence, etc.

As used herein, the term “expression vector” refers to a vectorcomprising a region which encodes a gene product of interest, and isused for effecting the expression of a gene product in an intendedtarget cell. An expression vector also comprises control elementsoperatively linked to the encoding region to facilitate expression ofthe protein in the target. The combination of control elements and agene or genes to which they are operably linked for expression issometimes referred to as an “expression cassette,” a large number ofwhich are known and available in the art or can be readily constructedfrom components that are available in the art.

As used herein, the term “expression” refers to the transcription and/ortranslation of a coding sequence, e.g. an endogenous gene, aheterologous gene, in a cell.

As used herein, the terms “gene” or “coding sequence” refer to apolynucleotide sequence that encodes a gene product, and encompassesboth naturally occurring polynucleotide sequences and cDNA. A gene mayor may not include regions preceding and following the coding region,e.g. 5′ untranslated (5′ UTR) or “leader” sequences and 3′ UTR or“trailer” sequences, or intervening sequences (introns) betweenindividual coding segments (exons).

As used herein, the term “gene product” refers the desired expressionproduct of a polynucleotide sequence such as a polypeptide, peptide,protein or RNA including, for example, a ribozyme, short interfering RNA(siRNA), miRNA or small hairpin RNA (shRNA). The terms “polypeptide,”“peptide,” and “protein” are used interchangeably herein to refer topolymers of amino acids of any length. The terms also encompass an aminoacid polymer that has been modified; for example, disulfide bondformation, glycosylation, lipidation, phosphorylation, or conjugationwith a labeling component.

As used herein, the terms “operatively linked” or “operably linked”refers to a juxtaposition of genetic elements on a singlepolynucleotide, wherein the elements are in a relationship permittingthem to operate in the expected manner. For instance, a promoter isoperatively linked to a coding region if the promoter helps initiatetranscription of the coding sequence. There may be intervening residuesbetween the promoter and coding region so long as this functionalrelationship is maintained. The combination of control elements, e.g.promoter, enhancer(s), etc. and a gene or genes to which they areoperably linked for expression is sometimes referred to as an“expression cassette,” a large number of which are known and availablein the art or can be readily constructed from components that areavailable in the art.

By a “promoter” it is generally meant a DNA sequence that directs thebinding of RNA polymerase and thereby promotes RNA synthesis, i.e., aminimal sequence sufficient to direct transcription. Promoters andcorresponding protein or polypeptide expression may be ubiquitous,meaning strongly active in a wide range of cells, tissues and species orcell-type specific, tissue-specific, or species-specific. Promoters maybe “constitutive,” meaning continually active, or “inducible,” meaningthe promoter can be activated or deactivated by the presence or absenceof biotic or abiotic factors.

By an “enhancer” it is generally meant a cis-acting regulatory elementthat stimulates, i.e. promotes or enhances, transcription of an adjacentgenes. By a “silencer” it is meant a cis-acting regulatory element thatinhibits, i.e. reduces or suppresses, transcription of an adjacent gene,e.g. by actively interfering with general transcription factor assemblyor by inhibiting other regulatory elements, e.g. enhancers, associatedwith the gene. Enhancers can function (i.e., can be associated with acoding sequence) in either orientation, over distances of up to severalkilobase pairs (kb) from the coding sequence and from a positiondownstream of a transcribed region. Enhancer sequences influencepromoter-dependent gene expression and may be located in the 5′ or 3′regions of the native gene. Enhancer sequences may or may not becontiguous with the promoter sequence. Likewise, enhancer sequences mayor may not be immediately adjacent to the gene sequence. For example, anenhancer sequence may be several thousand basepairs from the promoterand/or gene sequence.

A “termination signal sequence” within the meaning of the invention maybe any genetic element that causes RNA polymerase to terminatetranscription, such as for example a polyadenylation signal sequence. Apolyadenylation signal sequence is a recognition region necessary forendonuclease cleavage of an RNA transcript that is followed by thepolyadenylation consensus sequence AATAAA. A polyadenylation signalsequence provides a “polyA site”, i.e. a site on a RNA transcript towhich adenine residues will be added by post-transcriptionalpolyadenylation.

The terms “identical” or percent “identity” in the context of two ormore nucleotide sequences that are the same or have a specifiedpercentage of amino acid residues or nucleotides that are the same, whencompared and aligned for maximum correspondence, as measured using oneof the sequence comparison algorithms described herein, e.g. theSmith-Waterman algorithm, or by visual inspection.

As used herein, the term “sequence identity” refers to the degree ofidentify between nucleotides in two or more aligned sequences, whenaligned using a sequence alignment program. The term “% homology” isused interchangeably herein with the term “% identity” herein and refersto the level of nucleic acid or amino acid sequence identity between twoor more aligned sequences, when aligned using a sequence alignmentprogram. For example, as used herein, 80% homology means the same thingas 80% sequence identity determined by a defined algorithm, andaccordingly a homologue of a given sequence has greater than 80%sequence identity over a length of the given sequence. Sequence identitymay be determined by aligning sequences using any of a number ofpublicly available alignment algorithm tools, e.g., the local homologyalgorithm of Smith & Waterman, Adv. Appl. Math. 2: 482 (1981), thehomology alignment algorithm of Needleman & Wunsch, J Mol. Biol. 48: 443(1970), the search for similarity method of Pearson & Lipman, Proc.Nat'l. Acad. Sci. USA 85: 2444 (1988), computerized implementations ofthese algorithms (GAP, BESTFIT, FASTA, and TFASTA in the WisconsinGenetics Software Package, Genetics Computer Group, 575 Science Dr.,Madison, Wis.), by the BLAST algorithm, Altschul et al., J Mol. Biol.215: 403-410 (1990), with software that is publicly available throughthe National Center for Biotechnology Information(www.ncbi.nlm.nih.gov/), or by visual inspection (see generally, Ausubelet al., infra).

The terms “complement” and “complementary” refer to two antiparallelnucleotide sequences capable of pairing with one another upon formationof hydrogen bonds between the complementary base residues in theantiparallel nucleotide sequences.

The term “native”, when used in the context of a polynucleotide orpolypeptide herein, refers to a polynucleotide or polypeptide sequencethat is found in nature; i.e., that is present in the genome of awild-type virus or cell.

The term “variant’, when used in the context of a polynucleotide orpolypeptide herein, refers to a mutants of a native polynucleotide orpolypeptide having less than 100% sequence identity with the nativesequence or any other native sequence. Such variants may comprise one ormore substitutions, deletions, or insertions in the corresponding nativegene or gene product sequence. The term “variant” also includesfragments of the native gene or gene product, and mutants thereof, e.g.fragments comprising one or more substitutions, deletions, or insertionsin the corresponding native gene or gene product fragment. In someembodiments, the variant retains a functional activity of the nativegene product, e.g. ligand binding, receptor binding, protein signaling,etc., as known in the art.

The term “fragment,” when referring to a recombinant protein orpolypeptide of the invention means a polypeptide having an amino acidsequence which is the same as part of, but not all of, the amino acidsequence of the corresponding full length protein or polypeptide, whichretains at least one of the functions or activities of the correspondingfull length protein or polypeptide. The fragment preferably includes atleast 20-100 contiguous amino acid residues of the full length proteinor polypeptide.

As used herein, the terms “biological activity” and “biologicallyactive” refer to the activity attributed to a particular gene product,e.g. RNA or protein, in a cell line in culture or in vivo. For example,the “biological activity” of an RNAi molecule refers to the ability ofthe molecule to inhibit the production of a polypeptide from a targetpolynucleotide sequence.

As used herein, the term “antagonist” refers a molecule that acts toinhibit the activity of a target molecule. Antagonists include bothstructural antagonists that inhibit the activity of the target moleculeby, for example, binding directly to the target or inactivating itsreceptor and functional antagonists, which, for example, decreaseproduction of the target in a biological system or increase productionof inhibitors of the target in a biological system.

The terms “administering” or “introducing”, as used herein refer tocontacting a cell, tissue, or subject with a vector for the purposes ofdelivering a polynucleotide to the cell or to cells and or organs of thesubject. Such administering or introducing may take place in vivo, invitro or ex vivo. A vector for expression of a gene product may beintroduced into a cell by transfection, which typically means insertionof heterologous DNA into a cell by physical means (e.g., calciumphosphate transfection, electroporation, microinjection or lipofection);infection, which typically refers to introduction by way of aninfectious agent, i.e. a virus; or transduction, which typically meansstable infection of a cell with a virus or the transfer of geneticmaterial from one microorganism to another by way of a viral agent(e.g., a bacteriophage).

“Transformation” or “transfection” as used herein refers to the deliveryof a heterologous DNA to the interior of a cell, e.g. a mammalian cell,an insect cell, a bacterial cell, etc. by a vector. A vector used to“transform” a cell may be a plasmid, minicircle DNA, or other vehicle.Typically, a cell is referred to as “transduced”, “infected”;“transfected” or “transformed” dependent on the means used foradministration, introduction or insertion of heterologous DNA (i.e., thevector) into the cell. The terms “transfected” and “transformed” areused interchangeably herein to refer to the introduction of heterologousDNA by non-viral methods, e.g. electroporation, calcium chloridetransfection, lipofection, etc., e.g. as when preparing the subjectviral vectors for use in the subject methods. The terms “transduced” and“infected” are used interchangeably herein to refer to introduction ofthe heterologous DNA to the cell in the context of a viral particle.

The term “host cell”, as used herein refers to a cell which has beentransduced, infected, transfected or transformed with a vector. Thevector may be a plasmid, a viral particle, a phage, etc. The cultureconditions, such as temperature, pH and the like, are those previouslyused with the host cell selected for expression, and will be apparent tothose skilled in the art. It will be appreciated that the term “hostcell” refers to the original transduced, infected, transfected ortransformed cell and progeny thereof.

As used herein, a “therapeutic” gene refers to a gene that, whenexpressed, confers a beneficial effect on the cell or tissue in which itis present, or on a mammal in which the gene is expressed. Examples ofbeneficial effects include amelioration of a sign or symptom of acondition or disease, prevention or inhibition of a condition ordisease, or conferral of a desired characteristic. Therapeutic genesinclude genes that correct a genetic deficiency in a cell or mammal.

The terms “treatment”, “treating” and the like are used herein togenerally mean obtaining a desired pharmacologic and/or physiologiceffect. The effect may be prophylactic in terms of completely orpartially preventing a disease or symptom thereof, e.g. reducing thelikelihood that the disease or symptom thereof occurs in the subject,and/or may be therapeutic in terms of a partial or complete cure for adisease and/or adverse effect attributable to the disease. “Treatment”as used herein covers any treatment of a disease in a mammal, andincludes: (a) preventing the disease from occurring in a subject whichmay be predisposed to the disease but has not yet been diagnosed ashaving it; (b) inhibiting the disease, i.e., arresting its development;or (c) relieving the disease, i.e., causing regression of the disease.The therapeutic agent may be administered before, during or after theonset of disease or injury. The treatment of ongoing disease, where thetreatment stabilizes or reduces the undesirable clinical symptoms of thepatient, is of particular interest. Such treatment is desirablyperformed prior to complete loss of function in the affected tissues.The subject therapy will desirably be administered during thesymptomatic stage of the disease, and in some cases after thesymptomatic stage of the disease.

The terms “individual,” “subject,” “host,” and “patient,” are usedinterchangeably herein and refer to any mammalian subject for whomdiagnosis, treatment, or therapy is desired, including, but not limitedto, human and non-human primates, including simians and humans;mammalian sport animals (e.g., horses); mammalian farm animals (e.g.,sheep, goats, etc.); mammalian pets (dogs, cats, etc.); and rodents(e.g., mice, rats, etc.); particularly humans.

The terminology used herein is for the purpose of describing particularembodiments only and is not intended to be limiting of the invention. Asused herein, the singular forms “a”, “an” and “the” are intended toinclude the plural forms as well, unless the context clearly indicatesotherwise. Furthermore, to the extent that the terms “including”,“includes”, “having”, “has”, “with”, or variants thereof are used ineither the detailed description and/or the claims, such terms areintended to be inclusive in a manner similar to the term “comprising”.

By “comprising” it is meant that the recited elements are required in,for example, the composition, method, kit, etc., but other elements maybe included to form the, for example, composition, method, kit etc.within the scope of the claim. For example, an expression cassette“comprising” a gene encoding a therapeutic polypeptide operably linkedto a promoter is an expression cassette that may include other elementsin addition to the gene and promoter, e.g. poly-adenylation sequence,enhancer elements, other genes, linker domains, etc.

By “consisting essentially of”, it is meant a limitation of the scope ofthe, for example, composition, method, kit, etc., described to thespecified materials or steps that do not materially affect the basic andnovel characteristic(s) of the, for example, composition, method, kit,etc. For example, an expression cassette “consisting essentially of” agene encoding a therapeutic polypeptide operably linked to a promoterand a polyadenylation sequence may include additional sequences, e.g.linker sequences, so long as they do not materially affect thetranscription or translation of the gene. As another example, a variantpolypeptide fragment “consisting essentially of” a recited sequence hasthe amino acid sequence of the recited sequence plus or minus about 10amino acid residues at the boundaries of the sequence based upon thefull length naïve polypeptide from which it was derived, e.g. 10, 9, 8,7, 6, 5, 4, 3, 2 or 1 residue less than the recited bounding amino acidresidue, or 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 residues more than therecited bounding amino acid residue.

By “consisting of”, it is meant the exclusion from the composition,method, or kit of any element, step, or ingredient not specified in theclaim. For example, an expression cassette “consisting of” a geneencoding a therapeutic polypeptide operably linked to a promoter and apolyadenylation sequence consists only of the promoter, polynucleotidesequence encoding the therapeutic polypeptide, and polyadenlyationsequence. As another example, a polypeptide “consisting of” a recitedsequence contains only the recited amino acid sequence.

The term “about” or “approximately” means within an acceptable errorrange for the particular value as determined by one of ordinary skill inthe art, which will depend in part on how the value is measured ordetermined, i.e., the limitations of the measurement system. Forexample, “about” can mean within 1 or more than 1 standard deviation,per the practice in the art. Alternatively, “about” can mean a range ofup to 20%, preferably up to 10%, more preferably up to 5%, and morepreferably still up to 1% of a given value. Alternatively, particularlywith respect to biological systems or processes, the term can meanwithin an order of magnitude, preferably within 5-fold, and morepreferably within 2-fold, of a value. Where particular values aredescribed in the application and claims, unless otherwise stated theterm “about” meaning within an acceptable error range for the particularvalue should be assumed.

Methods and Compositions

In some aspects of the invention, methods and compositions are providedfor delivering a polynucleotide to cone photoreceptors. As discussedabove, cone photoreceptors, referred to interchangeably herein as “conecells”, “retinal cones”, and most simply, “cones,” are one of twosubtypes of photoreceptor cells in the retina of the eye, the otherbeing rod photoreceptors. Cone photoreceptors may be readilydistinguished from rod photoreceptors by a number of physical,biochemical, and functional characteristics. For example, conephotoreceptors comprise an outer segment region that is shaped like acone, whereas rod photoreceptors comprise an outer segment that isshaped like a rod. Cone photoreceptors express a number of proteins thatare not expressed by rod photoreceptors, including, e.g., L-opsin(OPN1LW, the nucleic acid and amino acid sequences for which may befound at GenBank Accession No:

NM 020061.5), M-opsin (OPN1MW, the nucleic acid and amino acid sequencesfor which may be found at GenBank Accession No: NM_000513.2), or S-opsin(OPN1SW, the nucleic acid and amino acid sequences for which may befound at GenBank Accession No: NM_001708.2); whereas rod photoreceptorsexpress a number of proteins that are not expressed by conephotoreceptors, e.g. rhodopsin (RHO, the nucleic acid and amino acidsequences for which may be found at GenBank Accession No: NM_000539.3)and rod-derived cone viability factor (RDCVF, also known as NXNL1, thenucleic acid and amino acid sequences for which may be found at GenBankAccession No: NM_138454.1). Functionally, cone photoreceptors differfrom rod photoreceptors in that cone photoreceptors are responsible forcolor vision and function best in relatively bright light, whereas rodphotoreceptors support vision at low light levels and function best indim light; cones and rods can be distinguished based on this differenceusing an electroretinogram (ERG) or color ERG (cERG). Finally, conephotoreceptors may be distinguished from rod photoreceptors by theirlocation in the retina. As discussed above, the vast majority of conephotoreceptors—all of them L- and M-cone photoreceptors—are denselypacked in a 1.5 mm depression located in the center of the macula of theretina, called the fovea centralis, with the remaining L- and M-conephotoreceptors and the S-cone photoreceptors scattered in the parafovea,the perifovea, and the peripheral retina. In contrast, rodphotoreceptors are excluded from the foveola and are poorly representedin the fovea, instead being primarily found in the parafovea, theperifovea, and the peripheral retina.

As discussed above, prior to the present disclosure, it was commonunderstanding in the art that cone photoreceptors—and more particularly,the L- and M-cone photoreceptors in the fovea—were resistant totransduction by AAV delivered from the vitreous. However, asdemonstrated by the working examples herein, foveal cones can, in fact,be transduced by intravitreally delivery using the methods andcompositions of the present disclosure. In some embodiments, the conephotoreceptors that are transduced by the subject methods andcompositions reside anywhere in the retina, i.e. the macula (the fovealcentralis, the parafovea, the perifovea), or the periphery. In someembodiments, the cone photoreceptors reside in the fovea centralis. Incertain embodiments, the cone photoreceptors are foveal cones, that is,they are L- or M-cones that reside within the fovea, this being theregion of the fovea centralis spanning from about 0.175 mm from thecenter of the fovea centralis to about 0.75 mm from the center of thefovea centralis.

rAAV Virions

In practicing the subject methods, the polynucleotide of interest isdelivered to cone photoreceptors by injecting into the vitreous of theeye a recombinant viral particle comprising the polynucleotide ofinterest as a heterologous sequence within its genome. In someinstances, the recombinant viral particles are recombinantadeno-associated virus (rAAV) particles. In some embodiments, the rAAVare of a wild-type serotype; that is, they comprise a viral capsid thatconsists of viral capsid proteins that occur in nature. In otherembodiments, the rAAV are an AAV serotype variant, i.e., they comprise avariant AAV capsid protein, that is, an AAV capsid protein thatcomprises at least one amino acid difference relative to a correspondingparental AAV capsid protein, e.g. a wild type AAV capsid protein, anddoes not consist of an amino acid sequence present in a naturallyoccurring AAV capsid protein.

As demonstrated in the working examples of the present application, rAAVvirions comprising a variant AAV capsid protein comprising at least oneamino acid difference in the GH loop, or more particularly, in subloopIV of the GH loop, demonstrate an increased infectivity of conephotoreceptors relative to rAAV virions comprising wild type AAV capsidprotein when delivered intravitreally. By “increased infectivity,” it ismeant that the variant rAAV virion is better able to transduce thetarget cell than the wild type AAV capsid protein. Improvements in theability of an AAV to transduce a cell can be observed by observing morepolynucleotide being delivered to each cell and more cells beingtransduced in a tissue, resulting in an increase in the amount ofpolynucleotide delivered to each cell and to the tissue. Accordingly, insome aspects of the invention, methods are provided for the improveddelivery of a polynucleotide of interest to cone photoreceptors, theimprovement comprising delivering to the vitreous of the eye aneffective amount of a rAAV variant, the rAAV variant comprising i) avariant AAV capsid protein that comprises at least one amino aciddifference relative to a corresponding parental AAV capsid protein, e.g.a wild type AAV capsid protein, and does not consist of an amino acidsequence present in a naturally occurring AAV capsid protein, and ii)the polynucleotide of interest as a heterologous sequence within theviral genome.

Of particular interest in the subject disclosure are rAAV variants thatcomprise at least one amino acid difference in the GH loop, or “loopIV”, of an AAV capsid protein relative to a corresponding parental AAVcapsid protein. By the GH loop, or loop IV, it is meant the loop createdbetween the G and H strands of the jelly-roll β-barrel of the AAV capsidprotein VP1, as described in, e.g., Xie et al. (2002) PNAS99(16):10405-10410, van Vliet et al. (2006) Mol. Ther. 14:809; Padron etal. (2005) J. Virol. 79:5047; and Shen et al. (2007) Mol. Ther. 15:1955.In some instances, the at least one amino acid difference is withinsubloop 4 of the GH loop, i.e., the solvent-accessible portion of the GHloop, consisting essentially of about amino acids 571-612 of AAV1 VP1(SEQ ID NO:1), about amino acids 570-611 of AAV2 VP1 (SEQ ID NO:2),about amino acids 571-612 of AAV3 VP1 (SEQ ID NO:3), about amino acids569-610 of AAV4 VP1 (SEQ ID NO:4), about amino acids 560-601 of AAV5 VP1(SEQ ID NO:5), about amino acids 571 to 612 of AAV6 VP1 (SEQ ID NO:6),about amino acids 572 to 613 of AAV7 VP1 (SEQ ID NO:7), about aminoacids 573 to 614 of AAV8 VP1 (SEQ ID NO:8), about amino acids 571 to 612of AAV9 VP1 (SEQ ID NO:9), about amino acids 573 to 614 of AAV10 VP1(SEQ ID NO:10); or about the corresponding amino acid range of a variantthereof. In certain instances, the at least one amino acid difference iswithin the range of amino acids consisting essentially of amino acids581-596 of AAV1 VP1, 580-595 of AAV2 VP1, 581-596 of AAV3 VP1, 579-594of AAV4, 570-585 of AAV5 VP1, 581-596 of AAV6 VP1, 582-597 of AAV7 VP1,583-598 of AAV8 VP1, 581-596 of AAV9 VP1, 583-598 of AAV10 VP1, orwithin the corresponding amino acid range of a variant thereof. Thoseskilled in the art would know, based on a comparison of the amino acidsequences of capsid proteins of various AAV serotypes, where the aminoacids “corresponding to amino acids 570-611 of VP1 from AAV2”, forexample, would be in a capsid protein of any given AAV serotype.

In some embodiments, the at least one amino acid difference is aninsertion of a peptide between two amino acids in the GH loop of the AAVcapsid protein, e.g. between about amino acids 571-612 of AAV1 VP1 (SEQID NO:1), about amino acids 570-611 of AAV2 VP1 (SEQ ID NO:2), aboutamino acids 571-612 of AAV3 VP1 (SEQ ID NO:3), about amino acids 569-610of AAV4 VP1 (SEQ ID NO:4), about amino acids 560-601 of AAV5 VP1 (SEQ IDNO:5), about amino acids 571 to 612 of AAV6 VP1 (SEQ ID NO:6), aboutamino acids 572 to 613 of AAV7 VP1 (SEQ ID NO:7), about amino acids 573to 614 of AAV8 VP1 (SEQ ID NO:8), about amino acids 571 to 612 of AAV9VP1 (SEQ ID NO:9), about amino acids 573 to 614 of AAV10 VP1 (SEQ IDNO:10); or about the corresponding amino acid range of a variantthereof; for example, between two amino acids within amino acids 581-596of AAV1 VP1, 580-595 of AAV2 VP1, 581-596 of AAV3 VP1, 579-594 of AAV4,570-585 of AAV5 VP1, 581-596 of AAV6 VP1, 582-597 of AAV7 VP1, 583-598of AAV8 VP1, 581-596 of AAV9 VP1, 583-598 of AAV10 VP1, or within thecorresponding amino acid range of a variant thereof. For example, theinsertion site can be between amino acids 580 and 581, amino acids 581and 582, amino acids 582 and 583, amino acids 583 and 584, amino acids584 and 585, amino acids 585 and 586, amino acids 586 and 587, aminoacids 587 and 588, amino acids 588 and 589, amino acids 589 and 590,amino acids 590 and 591, amino acids 591 and 592, amino acids 592 and593, amino acids 593 and 594, or amino acids 594 and 595 of AAV2 VP1, orthe corresponding amino acids in another AAV VP1 or variant thereof.

Of particular interest in some embodiments of the present disclosure arethe rAAV variants comprising a peptide insertion as disclosed in PCTPublication No. WO 2012/145601, the full disclosure of which isincorporated herein by reference. These rAAV variants comprise a peptideinsert having 5 to 11 amino acids in length, that is, the insertedpeptide comprises 5 amino acids, 6 amino acids, 7 amino acids, 8 aminoacids, 9 amino acids, 10 amino acids, or 11 amino acids.

One exemplary peptide of particular interest is a peptide of Formula I:

(SEQ ID NO: 20) Y₁Y₂X₁X₂X₃X₄X₅X₆X₇Y₃Y₄where:each of Y1-Y4, if present, is independently selected from Ala, Leu, Gly,Ser, and Thr;X1, if present, is selected from Leu, Asn, and Lys;X2 is selected from Gly, Glu, Ala, and Asp;X3 is selected from Glu, Thr, Gly, and Pro;X4 is selected from Thr, Ile, Gln, and Lys;X5 is selected from Thr and Ala;X6 is selected from Arg, Asn, and Thr;X7, if present, is selected from Pro and Asn.In certain embodiments, X1 and/or X7 is absent.

A second exemplary peptide of particular interest is a peptide ofFormula II:

(SEQ DI NO: 21) Y₁Y₂X₁X₂X₃X₄X₅X₆X₇Y₃Y₄where:each of Y1-Y4, if present, is independently selected from Ala, Leu, Gly,Ser, and Thr;each of X1-X4 is any amino acid;

X5 is Thr X6 is Arg; and X7 is Pro.

In certain embodiments, any one or more of Y1-Y4 are absent.

A third exemplary peptide of particular interest is a peptide of FormulaIII:

(SEQ ID NO: 22) Y₁Y₂X₁X₂X₃X₄X₅X₆X₇Y₃Y₄where:each of Y1-Y4, if present, is independently selected from Ala, Leu, Gly,Ser, and Thr;X1, if present, is selected from Leu and Asn;X2, if present, is selected from Gly and Glu;X3 is selected from Glu and Thr;X4 is selected from Thr and Ile;

X5 is Thr; X6 is Arg; and X7 is Pro.

In certain embodiments, any one or more of Y1-Y4, X1 and X2 are absent.

A fourth exemplary peptide of particular interest is a peptide ofFormula IV:

(SEQ ID NO: 23) Y1Y2X1X2X3X4X5X6X7Y3Y4where:each of Y1-Y4, if present, is independently selected from Ala, Leu, Gly,Ser, and Thr;X1, if present, is selected from Leu, Asn, Arg, Ala, Ser, and Lys;X2 is selected from Gly, Glu, Ala, Val, Thr, and Asp;X3 is selected from Glu, Thr, Gly, Asp, or Pro;X4 is selected from Thr, Ile, Gly, Lys, Asp, and Gln;X5 is selected from Thr, Ser, Val, and Ala;X6 is selected from Arg, Val, Lys, Pro, Thr, and Asn; andX7 is selected from Pro, Gly, Phe, Asn, and Arg.In certain embodiments, any one or more of Y1-Y4 and X1 are absent.

Exemplary insertion peptides of particular interest having theseformulas include peptides comprising the sequence LGETTRP (SEQ ID NO:11)and NETITRP (SEQ ID NO:12), or variants thereof. In some cases, theinsertion peptide has from 1 to 4 spacer amino acids (Y1-Y4) at theamino terminus and/or at the carboxyl terminus. Suitable spacer aminoacids include, but are not limited to, leucine, alanine, glycine, andserine. For example, in some cases, an insertion peptide has the aminoacid sequence: LALGETTRPA (SEQ ID NO:13); LANETITRPA (SEQ ID NO:14), Asanother example, in some cases, the insertion peptide has the amino acidsequence AALGETTRPA (SEQ ID NO:15) or AANETITRPA (SEQ ID NO:16), As yetanother example, in some cases, an insertion peptide has the amino acidsequence GLGETTRPA (SEQ ID NO:17) or GNETITRPA (SEQ ID NO:18).

In some embodiments, a subject rAAV virion capsid does not include anyamino acid substitutions, insertions, or deletions, other than aninsertion of from about 5 to 11 amino acids in the GH loop or subregionthereof relative to a corresponding parental AAV capsid protein. Inother embodiments, a subject rAAV virion capsid may include from 1 toabout 25 amino acid insertions, deletions, or substitutions, compared tothe parental AAV capsid protein, in addition to an insertion of fromabout 5 to 11 amino acids in the GH loop or subregion thereof asdescribed above. For example, a number of amino acid sequencealterations have been disclosed in the art, any of which may be includedin the subject rAAV. In some embodiments, a subject rAAV virion capsidis a chimeric capsid, e.g., the capsid comprises a portion of an AAVcapsid of a first AAV serotype and a portion of an AAV capsid of asecond serotype; and comprises an insertion of from about 5 amino acidsto about 11 amino acids in the GH loop or subregion thereof relative toa corresponding parental AAV capsid protein.

In some embodiments, a subject rAAV virion comprises a capsid proteincomprising an amino acid sequence having a sequence identity of 80% ormore to the VP1 capsid protein of the corresponding parental capsidprotein, e.g. 85% or more, 90% or more, 95% or more or 97 C % identityor more to the corresponding parental capsid protein and an insertion offrom about 5 to 11 amino acids in the GH loop or subregion thereofrelative to a corresponding parental AAV capsid protein. For example asequence identity of 80% or more to the 7m8 VP1 sequence described inSEQ ID NO:19, e.g. 85% identity or more, 90% identity or more, or 95%identity or more to the 7m8 VP1 sequence, in some instances 97% identityor more, 98% identity or more, or at least about 99% sequence identityto the amino acid sequence provided in SEQ ID NO:19.

rAAV variants that are encompassed by the subject compositions and thatfind use in the subject methods may be readily validated as such bydetermining the efficacy by which they transduce cone photoreceptors,e.g. foveal cone photoreceptors. For example, viral particles may becreated comprising an AAV viral genome comprising an expression cassettecomprising GFP operably linked to a cone promoter as known in the art,packaged into the subject rAAV, and the viral particles injected intothe vitreous of a mammalian eye, e.g. the eye of a mouse, rat, rabbit,gerbil, hamster, squirrel, or primate, e.g. non-human primate. rAAVvirions encompassed by the present disclosure will typically exhibit atleast a 2-fold, at least a 5-fold, at least a 10-fold, at least a15-fold, at least a 20-fold, at least a 25-fold, at least a 50-fold, insome instances, more than 50-fold, e.g. at least a 60-fold, at least a70-fold, at least an 80-fold, at least a 90-fold, for example, a100-fold increased infectivity of cone photoreceptors or more whenadministered via intravitreal injection as compared to the infectivityof cone photoreceptors by an AAV virion comprising the correspondingparental AAV capsid protein. Put another way, rAAV virions suitable foruse in the subject methods will infect at least 10-fold more, at least15-fold more, at least 20-fold more, at least 50-fold more, in someinstances more than 50-fold more cone photoreceptors, e.g. at least60-fold, at least 70-fold, at least 80-fold, at least 90-fold, forexample, a 100-fold more cone photoreceptors than AAV virions comprisingthe corresponding parental AAV capsid protein.

In some embodiments, the method may further comprise the step ofdetecting the presence of the delivered polynucleotide in the conephotoreceptor. Any convenient method may be employed for detecting thepresence of the polynucleotide. For example, the polynucleotide may bedetecting using, e.g., PCR, Next Gen sequencing, and the like, or theexpression of a gene product encoded by the polynucleotide may bedetected by, e.g., RT-PCR, Northern blot, RNAse protection, Westernblot, ELISA, immunohistochemistry, and the like. These methods areparticularly suited to preclinical studies. In clinical studies, in maybe preferably to detect the presence of the polynucleotide by detectingthe presence of a functional gene product, that is, by detecting theimpact of the gene product on the viability or function of the conephotoreceptor in the subject. For example, if the gene product encodedby the polynucleotide improves the viability of the cone photoreceptor,an improvement in viability of the cone photoreceptor may be detectedby, e.g., fundus photography, Optical coherence tomography (OCT),Adaptive Optics (AO), and the like, as a way of detecting the presenceof the polynucleotide. If the gene product encoded by the polynucleotidealters the activity of the cone photoreceptor, the modified activity ofthe cone photoreceptor may be detected by, e.g., electroretinogram (ERG)and color ERG (cERG); color vision tests such as pseudoisochromaticplates (Ishihara plates, Hardy-Rand-Ritter polychromatic plates), theFarnsworth-Munsell 100 hue test, the Farnsworth's panel D-15, the Cityuniversity test, Kollner's rule, and the like; and visual acuity testssuch as the ETDRS letters test, Snellen visual acuity test, and thelike, as a way of detecting the presence of the deliveredpolynucleotide.

As discussed above, in some embodiments, the polynucleotide that isdelivered by the subject compositions and methods is expressed by thecone photoreceptor to which it is delivered. In other words, in someaspects of the invention, methods are provided for expressing a geneproduct in a cone photoreceptor, the methods comprising delivering tothe cone photoreceptor a polynucleotide that encodes the gene product ofinterest. As will be well understood by the ordinarily skilled artisan,expression by a cone cell of a polynucleotide of interest typicallyrequires that the polynucleotide of interest be operably linked to apromoter. As will also be appreciated by the ordinarily skilled artisan,there are a number of ways in which this can be achieved. For example,the polynucleotide may be delivered to the host cell, i.e. the conephotoreceptor, operatively linked to a promoter. In other words, theviral genome comprising the polynucleotide of interest also comprises apromoter, wherein the promoter is operably linked to the polynucleotideto form an expression cassette. As another example, the polynucleotidemay be delivered to the host cell i.e. the cone photoreceptor, flankedby sequences that promoter the integration of the polynucleotide intothe host genome. In other words, the viral genome comprising thepolynucleotide of interest comprises sequences flanking thepolynucleotide of interest that are homologous to sequences flanking the3′ end of a host cell promoter and promote the recombination of thepolynucleotide of interest into the host genome such that it is operablylinked to the host cell promoter. Other arrangements of the recombinantviral genome that may be employed to ensure the expression of thepolynucleotide of interest will be readily envisioned by the ordinarilyskilled artisan; see, for example, US Application Publication No.2013/0280222, the full disclosure of which is incorporated herein byreference.

Accordingly, in some instances, the viral genome comprised by the rAAVcomprises a promoter operably linked to the polynucleotide of interest.In some instances, the promoter is a ubiquitous promoter, i.e., it is apromoter that is active in a wide range of cells, tissues and species.In other instances, the promoter is a cone promoter. By a cone promoterit is meant a promoter that is active in cone photoreceptors, i.e., thatpromotes the expression in cone photoreceptors of a polynucleotide towhich it is operably linked. Non-limiting examples of cone promotersthat find use in the subject compositions include the pMNTC promoter asdisclosed in U.S. Provisional Application Nos. 61/954,330 and62/127,185; the pR2.1 promoter or variants thereof (e.g. pR1.7, pR1.5,pR1.1, etc.) as disclosed in, e.g., US Application No. 2013/0317091; orthe synthetic IRBP/GNAT2 promoter as disclosed in US Application No.2014/0275231; the full disclosures of which are incorporated herein byreference. In other instances, the viral genome comprised by the rAAVcomprises two sequences having homology to a target integration site inthe host genome, a first sequence that is homologous to the region 5′ ofthe integration site and located 5′ to the polynucleotide on the viralgenome, and a second sequence that is homologous to the region 3′ of theintegration site and located 3′ to the polynucleotide on the viralgenome, wherein the target integration site is 3′ to and operably linkedto a host promoter, e.g. a cone promoter, e.g. an L-opsin promoter, anM-opsin promoter.

In some embodiments, transduction is enhanced relative to expression asobserved when a wild type or other parental capsid is employed. Byenhanced, it is meant transduction that is elevated, increased, oraugmented for example, at least 2-fold, at least 5-fold, at least10-fold, at least 15-fold, at least 20-fold, at least 25-fold, at least50-fold, in some instances, more than 50-fold, e.g. at least 60-fold, atleast 70-fold, at least 80-fold, at least 90-fold, for example, 100-foldin a subject's cone photoreceptors over levels that would be observedusing a wild type or other parental capsid protein, and usually to anamount to have an impact on cone viability and/or function, e.g. toprovide a therapeutic benefit to the subject.

Enhanced transduction of cone cells by the subject variant rAAVs isexpected to result in enhanced expression of polynucleotides, e.g.,expression cassettes, being delivered to those cells by the variantrAAV. Enhanced expression of a polynucleotide by the rAAVs of thesubject disclosure may be observed in a number of ways. For example,enhanced expression may be observed by detecting the expression of thepolynucleotide following contact of the variant rAAV to the cone cellssooner, e.g. 7 days sooner, 2 weeks sooner, 3 weeks sooner, 4 weekssooner, 8 weeks sooner, 12 weeks sooner, or more, than expression wouldbe detected if the polynucleotide were delivered by the parental rAAV.Enhanced expression may also be observed as an increase in the amount ofgene product per cell. For example, there may be a 2-fold increase ormore, e.g. a 3-fold increase or more, a 4-fold increase or more, a5-fold increase or more, or a 10-fold increase or more in the amount ofgene product per cone cell. Enhanced expression may also be observed asan increase in the number of cone cells that express detectable levelsof the polynucleotide carried by the variant rAAV. For example, theremay be a 2-fold increase or more, e.g. a 3-fold increase or more, a4-fold increase or more, a 5-fold increase or more, or a 10-foldincrease or more in the number of cone cells that express detectablelevels of the polynucleotide. As another example, the polynucleotide ofthe present invention may promote detectable levels of thepolynucleotide in a greater percentage of cells as compared to aparental rAAV; for example, where a parental rAAV may promote detectablelevels of polynucleotide expression in, for example, less than 5% of thecone cells in a certain region, the rAAV of the present inventionpromotes detectable levels of expression in 5% or more of the cone cellsin that region; e.g. 10% or more, 15% or more, 20% or more, 25% or more,30% or more, 35% or more, 40% or more, or 45% or more, in some instances50% or more, 55% or more; 60% or more, 65% or more, 70% or more, or 75%or more, for example 80% or more, 85% or more, 90% or more, or 95% ormore of the cone cells that are contacted, will express detectablelevels of gene product. Enhanced expression may also be observed as analteration in the viability and/or function of the cone cells, e.g. asmeasured using assessment tools such as fundus photography, OCT,adaptive optics, cERG, color vision tests, visual acuity tests, and thelike, as known in the art and as described herein.

In some embodiments, the method may further comprise the step ofdetecting the expression of the polynucleotide in the conephotoreceptor. In such embodiments, any convenient method as known inthe art or described herein may be employed for detecting the expressionof the polynucleotide, including, for example, detecting the geneproduct, i.e., the encoded RNA or protein, e.g., by RT-PCR, Northernblot, RNAse protection, Western blot, ELISA, immunohistochemistry, andthe like; detecting the impact of the gene product on the viability ofthe cone photoreceptor, e.g., by fundus photography, Optical coherencetomography (OCT), Adaptive Optics (AO); or detecting the impact of thegene product on cone function, e.g. electroretinography (ERG), colorvision tests, visual acuity tests, etc., any of which may be employed inthe subject methods.

rAAV virions comprising the polynucleotide of interest of the presentdisclosure may be produced using any convenient methodologies, AAVpackaging cells, and packaging technology as known to those of skill inthe art. For example, an AAV expression vector (that is, a plasmidcomprising the rAAV genome as well as elements useful for the cloning ofthe genomic elements in, e.g. bacteria, e.g. origin of replication,selectable marker, etc.) may be transfected into mammalian producercells. Also transfected into the mammalian producer cells is an AAVhelper construct, i.e. a plasmid comprising AAV REP and CAP codingregions that can be expressed in the producer cell, which complement AAVhelper functions absent from the AAV expression vector. Thedually-transfected producer cells are then infected by a helper virus,e.g. adenovirus, or transfected with a plasmid comprising helper virusaccessory genes that promote AAV vector replication, e.g., regions VA,E2A, E4, so as to promote efficient rAAV virus production. The producercells are then cultured to produce rAAV, and AAV vectors are purifiedand formulated using standard techniques known in the art.

As another example, an AAV expression vector may be packaged as abaculovirus and introduced into insect producer cells, e.g. Sf9 cells.Also introduced into the insect cells by another baculovirus are the AAVREP and CAP genes. Baculovirus-being a virus—comprises the genesencoding the accessory functions necessary for efficient rAAV virusproduction. Accordingly, upon infection of the insect cells by the twobaculoviruses, the producer cells can be cultured to produce rAAV, andAAV vectors purified and formulated using standard techniques known inthe art.

Examples of these and other methods may be found in, for example, U.S.Pat. Nos. 5,436,146; 5,753,500, 6,040,183, 6,093,570 and 6,548,286,expressly incorporated by reference herein in their entirety. Furthercompositions and methods for packaging are described in Wang et al. (US2002/0168342), also incorporated by reference herein in its entirety.

Any convenient host cells used in the art for producing rAAV virions maybe employed in the production of the subject vectors, including, forexample, mammalian cells, insect cells, microorganisms and yeast, e.g.SF-9, 293, A549, HeLa cells, etc. In some instances, the host cells arepackaging cells in which the AAV rep and cap genes are stably maintainedin the host cell. In some instances, the host cells are producer cellsin which the AAV vector genome is stably maintained and packaged.

Pharmaceutical Compositions and Unit Dosages

In some embodiments, e.g. gene therapy uses, it will be desirable toformulate the subject rAAV as a pharmaceutical composition. In certainembodiments, a pharmaceutical composition comprises a vector or virion(e.g., rAAV) described herein and one or more pharmaceuticallyacceptable carriers, diluents or excipients. Pharmaceutical compositionssuitable for use include sterile aqueous solutions or dispersions andsterile powders for the extemporaneous preparation of sterile injectablesolutions or dispersion. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, orphosphate buffered saline (PBS). In all cases, the composition must besterile and should be fluid to the extent that easy syringabilityexists. It must be stable under the conditions of manufacture andstorage and must be preserved against the contaminating action ofmicroorganisms such as bacteria and fungi. The carrier can be a solventor dispersion medium containing, for example, water, ethanol, polyol(for example, glycerol, propylene glycol, and liquid polyethyleneglycol, and the like), and suitable mixtures thereof. The properfluidity can be maintained, for example, by the use of a coating such aslecithin, by the maintenance of the required particle size in the caseof dispersion and by the use of surfactants. Prevention of the action ofmicroorganisms can be achieved by various antibacterial and antifungalagents, for example, parabens, chlorobutanol, phenol, ascorbic acid,thimerosal, and the like. In many cases, it will be preferable toinclude isotonic agents, for example, sugars, polyalcohols such asmanitol, sorbitol, sodium chloride in the composition. Prolongedabsorption of the internal compositions can be brought about byincluding in the composition an agent which delays absorption, forexample, aluminum monostearate and gelatin.

Sterile solutions can be prepared by incorporating the active compoundin the required amount in an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the active compound into a sterile vehicle that contains abasic dispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, methods of preparation are vacuum dryingand freeze-drying that yields a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof.

In one embodiment, active compounds are prepared with carriers that willprotect the compound against rapid elimination from the body, such as acontrolled release formulation, including implants and microencapsulateddelivery systems. Biodegradable, biocompatible polymers can be used,such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid,collagen, polyorthoesters, and polylactic acid. Methods for preparationof such formulations will be apparent to those skilled in the art. Thematerials can also be obtained commercially. Liposomal suspensions(including liposomes targeted to infected cells with monoclonalantibodies to viral antigens) can also be used as pharmaceuticallyacceptable carriers. These can be prepared according to methods known tothose skilled in the art, for example, as described in U.S. Pat. No.4,522,811.

The pharmaceutical compositions of the subject disclosure encompass anypharmaceutically acceptable salts, esters, or salts of such esters, orany other compound which, upon administration to an animal comprising ahuman, is capable of providing (directly or indirectly) the biologicallyactive metabolite or residue thereof. Accordingly, for example, thedisclosure is also drawn to prodrugs and pharmaceutically acceptablesalts of the compounds of the invention, pharmaceutically acceptablesalts of such prodrugs, and other bio-equivalents.

The term “prodrug” indicates a therapeutic agent that is prepared in aninactive form that is converted to an active form (i.e., drug) withinthe body or cells thereof by the action of endogenous enzymes or otherchemicals and/or conditions.

The term “pharmaceutically acceptable salt” refers to physiologicallyand pharmaceutically acceptable salts of the compounds of the invention:i.e., salts that retain the desired biological activity of the parentcompound and do not impart undesired toxicological effects thereto.

Pharmaceutically acceptable base addition salts are formed with metalsor amines, such as alkali and alkaline earth metals or organic amines.Metals used as cations comprise sodium, potassium, magnesium, calcium,and the like. Amines comprise N—N′-dibenzylethylenediamine,chloroprocaine, choline, diethanolamine, dicyclohexylamine,ethylenediamine, N-methylglucamine, and procaine (see, for example,Berge et al., “Pharmaceutical Salts,” J. Pharma Sci., 1977, 66, 119).The base addition salts of said acidic compounds are prepared bycontacting the free acid form with a sufficient amount of the desiredbase to produce the salt in the conventional manner. The free acid formmay be regenerated by contacting the salt form with an acid andisolating the free acid in the conventional manner. The free acid formsdiffer from their respective salt forms somewhat in certain physicalproperties such as solubility in polar solvents, but otherwise the saltsare equivalent to their respective free acid for purposes of the presentinvention.

As used herein, a “pharmaceutical addition salt” comprises apharmaceutically acceptable salt of an acid form of one of thecomponents of the compositions of the invention. These comprise organicor inorganic acid salts of the amines. Preferred acid salts are thehydrochlorides, acetates, salicylates, nitrates and phosphates. Othersuitable pharmaceutically acceptable salts are well known to thoseskilled in the art and comprise basic salts of a variety of inorganicand organic acids, such as, for example, with inorganic acids, such asfor example hydrochloric acid, hydrobromic acid, sulfuric acid orphosphoric acid; with organic carboxylic, sulfonic, sulfo or phosphoacids or N-substituted sulfamic acids, for example acetic acid,propionic acid, glycolic acid, succinic acid, maleic acid, hydroxymaleicacid, methylmaleic acid, fumaric acid, malic acid, tartaric acid, lacticacid, oxalic acid, gluconic acid, glucaric acid, glucuronic acid, citricacid, benzoic acid, cinnamic acid, mandelic acid, salicylic acid,4-aminosalicylic acid, 2-phenoxybenzoic acid, 2-acetoxybenzoic acid,embonic acid, nicotinic acid or isonicotinic acid; and with amino acids,such as the 20 alpha-amino acids involved in the synthesis of proteinsin Nature, for example glutamic acid or aspartic acid, and also withphenylacetic acid, methanesulfonic acid, ethanesulfonic acid,2-hydroxyethanesulfonic acid, ethane-1,2-disulfonic acid,benzenesulfonic acid, 4-methylbenzenesulfoic acid,naphthalene-2-sulfonic acid, naphthalene-1,5-disulfonic acid, 2- or3-phosphoglycerate, glucose-6-phosphate, N-cyclohexylsulfamic acid (withthe formation of cyclamates), or with other acid organic compounds, suchas ascorbic acid. Pharmaceutically acceptable salts of compounds mayalso be prepared with a pharmaceutically acceptable cation. Suitablepharmaceutically acceptable cations are well known to those skilled inthe art and comprise alkaline, alkaline earth, ammonium and quaternaryammonium cations. Carbonates or hydrogen carbonates are also possible.For oligonucleotides, preferred examples of pharmaceutically acceptablesalts comprise but are not limited to: (I) salts formed with cationssuch as sodium, potassium, ammonium, magnesium, calcium, polyamides suchas spermine and spermidine, and the like; (II) acid addition saltsformed with inorganic acids, for example hydrochloric acid, hydrobromicacid, sulfuric acid, phosphoric acid, nitric acid and the like; (III)salts formed with organic acids such as, for example, acetic acid,oxalic acid, tartaric acid, succinic acid, maleic acid, fumaric acid,gluconic acid, citric acid, malic acid, ascorbic acid, benzoic acid,tannic acid, palmitic acid, alginic acid, polyglutamic acid,napthalenesulfonic acid, methanesulfonic acid, p-toluenesulfonic acid,naphthalenedisulfonic acid, polygalacturonic acid, and the like; and(IV) salts formed from elemental anions such as chlorine, bromine, andiodine.

Pharmaceutical compositions of the present invention comprise, but arenot limited to, solutions, emulsions, and liposome-containingformulations. These compositions may be generated from a variety ofcomponents that comprise, but are not limited to, preformed liquids,self-emulsifying solids and self-emulsifying semisolids.

Certain compositions of the present invention also incorporate carriercompounds in the formulation. As used herein, “carrier compound” or“carrier” can refer to a nucleic acid, or analog thereof, which is inert(i.e., does not possess biological activity per se) but is recognized asa nucleic acid by in vivo processes that reduce the bioavailability of anucleic acid having biological activity by, for example, degrading thebiologically active nucleic acid or promoting its removal fromcirculation. The co-administration of a nucleic acid and a carriercompound, typically with an excess of the latter substance, can resultin a substantial reduction of the amount of nucleic acid recovered inthe liver, kidney or other extra circulatory reservoirs, presumably dueto competition between the carrier compound and the nucleic acid for acommon receptor. For example, the recovery of a partiallyphosphorothioate oligonucleotide in hepatic tissue can be reduced whenit is co-administered with polyinosinic acid, dextran sulphate,polycytidic acid or 4-acetamido-4′isothiocyano-stilbene-2,2′disulfonicacid (Miyao et al., Antisense Res. Dev., 1995, 5, 115-121; Takakura etal., Antisense & Nucl. Acid Drug Dev., 1996, 6, 177-183).

The subject recombinant AAV can be incorporated into pharmaceuticalcompositions for administration to mammalian patients, particularlyhumans. The virions can be formulated in nontoxic, inert,pharmaceutically acceptable aqueous carriers, preferably at a pH rangingfrom 3 to 8, more preferably ranging from 6 to 8. Such sterilecompositions will comprise the vector or virion containing the nucleicacid encoding the therapeutic molecule dissolved in an aqueous bufferhaving an acceptable pH upon reconstitution.

In some embodiments, the pharmaceutical composition provided hereincomprise a therapeutically effective amount of a vector or virion inadmixture with a pharmaceutically acceptable carrier and/or excipient,for example saline, phosphate buffered saline, phosphate and aminoacids, polymers, polyols, sugar, buffers, preservatives and otherproteins. Exemplary amino acids, polymers and sugars and the like areoctylphenoxy polyethoxy ethanol compounds, polyethylene glycolmonostearate compounds, polyoxyethylene sorbitan fatty acid esters,sucrose, fructose, dextrose, maltose, glucose, mannitol, dextran,sorbitol, inositol, galactitol, xylitol, lactose, trehalose, bovine orhuman serum albumin, citrate, acetate, Ringer's and Hank's solutions,cysteine, arginine, carnitine, alanine, glycine, lysine, valine,leucine, polyvinylpyrrolidone, polyethylene and glycol. Preferably, thisformulation is stable for at least six months at 4° C.

In some embodiments, the pharmaceutical composition provided hereincomprises a buffer, such as phosphate buffered saline (PBS) or sodiumphosphate/sodium sulfate, tris buffer, glycine buffer, sterile water andother buffers known to the ordinarily skilled artisan such as thosedescribed by Good et al. (1966) Biochemistry 5:467. The pH of the bufferin which the pharmaceutical composition comprising the tumor suppressorgene contained in the adenoviral vector delivery system, may be in therange of 6.5 to 7.75, preferably 7 to 7.5, and most preferably 7.2 to7.4.

In some embodiments, the pharmaceutical composition provided hereincomprises substances which increase the viscosity of the suspension,such as sodium carboxymethyl cellulose, sorbitol, or dextran, in theamount about 1-10 percent, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10percent.

The pharmaceutical compositions can be included in a container, pack, ordispenser together with instructions for administration.

In some instances, e.g. for administration intraocularly, orally, orparentally, it may be especially advantageous to formulate thepharmaceutical composition in dosage unit form for ease ofadministration and uniformity of dosage. Dosage unit form as used hereinrefers to physically discrete units suited as unitary dosages for thesubject to be treated; each unit containing a predetermined quantity ofactive compound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms of the invention are dictated by and directlydependent on the unique characteristics of the active compound and theparticular therapeutic effect to be achieved, and the limitationsinherent in the art of compounding such an active compound for thetreatment of individuals.

In some cases, the unit dose of the pharmaceutical composition of thedisclosure may be measured as pfu (plaque forming units). In some cases,the pfu of the unit dose of the pharmaceutical composition of thedisclosure may be about 1×10⁸ to about 5×10¹⁰ pfu. In some cases, thepfu of the unit dose of the pharmaceutical composition of the disclosureis at least about 1×10⁸, 2×10⁸, 3×10⁸, 4×10⁸, 5×10⁸, 6×10⁸, 7×10⁸,8×10⁸, 9×10⁸, 1×10⁹, 2×10⁹, 3×10⁹, 4×10⁹, 5×10⁹, 6×10⁹, 7×10⁹, 8×10⁹,9×10⁹, 1×10¹⁰, 2×10¹⁰, 3×10¹⁰, 4×10¹⁰, and 5×10¹⁰ pfu. In some cases,the pfu of the unit dose of the pharmaceutical composition of thedisclosure is at most about 1×10⁸, 2×10⁸, 3×10⁸, 4×10⁸, 5×10⁸, 6×10⁸,7×10⁸, 8×10⁸, 9×10⁸, 1×10⁹, 2×10⁹, 3×10⁹, 4×10⁹, 5×10⁹, 6×10⁹, 7×10⁹,8×10⁹, 9×10⁹, 1×10¹⁰, 2×10¹⁰, 3×10¹⁰, 4×10¹⁰, and 5×10¹⁰ pfu.

In some cases, the viral vector of the disclosure may be measured asvector genomes. In some cases, the unit dose of the pharmaceuticalcomposition of the disclosure is 1×10⁸ vector genomes or more, e.g.1×10⁹, 1×10¹⁰, 1×10¹¹, 1×10¹², or 1×10¹³ vector genomes or more, incertain instances, 1×10¹⁴ vector genomes or more, and usually no morethan 1×10¹⁵ vector genomes. In some embodiments, the unit dose of thepharmaceutical composition of the disclosure is at most about 1×10¹⁵vector genomes, e.g. 1×10¹⁴ vector genomes or less, for example 1×10¹³,1×10¹², 1×10¹¹, 1×10¹⁰, or 1×10⁹ vector genomes or less, in certaininstances 1×10⁸ vector genomes, and typically no less than 1×10⁸ vectorgenomes. In some cases, the unit dose of the pharmaceutical compositionof the disclosure is 1×10¹⁰ to 1×10¹¹ vector genomes. In some cases, theunit dose of the pharmaceutical composition of the disclosure is 1×10¹⁰to 3×10¹² vector genomes. In some cases, the unit dose of thepharmaceutical composition of the disclosure is 1×10⁹ to 3×10¹³ vectorgenomes. In some cases, the unit dose of the pharmaceutical compositionof the disclosure is 1×10⁸ to 3×10¹⁴ vector genomes.

In some cases, the unit dose of the pharmaceutical composition of thedisclosure may be measured using multiplicity of infection (MOI). Insome cases, MOI may refer to the ratio, or multiple of vector or viralgenomes to the cells to which the nucleic may be delivered. In somecases, the MOI may be 1×10⁶. In some cases, the MOI may be 1×10⁵-1×10⁷.In some cases, the MOI may be 1×10⁴-1×10⁸. In some cases, recombinantviruses of the disclosure are at least about 1×10¹, 1×10², 1×10³, 1×10⁴,1×10⁵, 1×10⁶, 1×10⁷, 1×10⁸, 1×10⁹, 1×10¹⁰, 1×10¹¹, 1×10¹², 1×10¹³,1×10¹⁴, 1×10¹⁵, 1×10¹⁶, 1×10¹⁷, and 1×10¹⁸ MOI. In some cases,recombinant viruses of this disclosure are 1×10⁸ to 3×10¹⁴ MOI. In somecases, recombinant viruses of the disclosure are at most about 1×10¹,1×10², 1×10³, 1×10⁴, 1×10⁵, 1×10⁶, 1×10⁷, 1×10⁸, 1×10⁹, 1×10¹⁰, 1×10¹¹,1×10¹², 1×10¹³, 1×10¹⁴, 1×10¹⁵, 1×10¹⁶, 1×10¹⁷, and 1×10¹⁸ MOI.

In some aspects, the pharmaceutical composition comprises about 1×10⁸ toabout 1×10¹⁵ recombinant viruses, about 1×10⁹ to about 1×10¹⁴recombinant viruses, about 1×10¹⁰ to about 1×10¹³ recombinant viruses,about 1×10¹⁰⁹ to about 3×10¹² recombinant viruses, or about 1×10¹¹ toabout 3×10¹² recombinant viruses.

Methods of Administration

The pharmaceutical composition of the present invention may beadministered to the eye of the subject by any convenient method, e.g.intraocularly, intravenously, intraperitoneally, etc. In some instances,the administration is intraocular, e.g. by intravitreal injection orsubretinal injection. The general methods for delivering a vector viaintravitreal injection or via subretinal injection may be illustrated bythe following brief outlines. These examples are merely meant toillustrate certain features of the methods, and are in no way meant tobe limiting.

In preferred embodiments, the subject rAAV is delivered intravitreally.For intravitreal administration, the vector can be delivered in the formof a suspension. Initially, topical anesthetic is applied to the surfaceof the eye followed by a topical antiseptic solution. The eye is heldopen, with or without instrumentation, and the vector is injectedthrough the sclera with a short, narrow, for example a 30 gauge needle,into the vitreous cavity of the eye of a subject under directobservation. Intravitreal administration is generally well tolerated. Atthe conclusion of the procedure, there is sometimes mild redness at theinjection site. There is occasional tenderness, but most patients do notreport any pain. No eye patch or eye shield is necessary after thisprocedure, and activities are not restricted. Sometimes, an antibioticeye drop is prescribed for several days to help prevent infection.

In some embodiments, the subject rAAV is delivered subretinally. Forsubretinal administration, the vector can be delivered in the form of asuspension injected subretinally under direct observation using anoperating microscope. This procedure may involve vitrectomy followed byinjection of vector suspension using a fine cannula through one or moresmall retinotomies into the subretinal space.

Briefly, an infusion cannula can be sutured in place to maintain anormal globe volume by infusion (of e.g. saline) throughout theoperation. A vitrectomy is performed using a cannula of appropriate boresize (for example 20 to 27 gauge), wherein the volume of vitreous gelthat is removed is replaced by infusion of saline or other isotonicsolution from the infusion cannula. The vitrectomy is advantageouslyperformed because (1) the removal of its cortex (the posterior hyaloidmembrane) facilitates penetration of the retina by the cannula; (2) itsremoval and replacement with fluid (e.g. saline) creates space toaccommodate the intraocular injection of vector, and (3) its controlledremoval reduces the possibility of retinal tears and unplanned retinaldetachment.

In practicing the subject methods, the subject rAAV virion is deliveredto the eye in an amount effective to deliver the polynucleotide ofinterest to 5% or more of the subject's cone photoreceptors, forexample, 10% or more, 20% or more, 30% or more, 40% or more, or 50% ormore of the subject's cone photoreceptors, e.g. 60% or more, 70% ormore, 80% or more, or 90% or more of the subject's cone photoreceptors,in some instance, 95% or more, 98% or more, or 100% of the subject'scone photoreceptors to provide therapeutic benefit to the subjectindividual. Put another way, following the administering, 5% or more ofthe subject's cone photoreceptors, e.g. 10% or more, 20% or more, 30% ormore, 40% or more, or 50% or more, in some instance 60% or more, 70% ormore, 80% or more, or 90% or more, e.g. 95%, 98%, or 100% of the cones,will comprise a sufficient amount of the polynucleotide of interest tohave an impact on cone viability and/or function, e.g. to treat orprevent a disorder. In some embodiments, the transduced conesphotoreceptors will be located throughout the retina. In someembodiments, the transduced cone photoreceptors will be cones in thefovea and foveola. In some embodiments, the transduced conephotoreceptors will be foveal cones, i.e. L- or M-cones located in thefovea.

Typically, an effective amount will be about 1×10⁸ vector genomes ormore of the subject rAAV, e.g. 1×10⁹, 1×10¹⁰, 1×10¹¹, 1×10¹², or 1×10¹³vector genomes or more, in certain instances, 1×10¹⁴ vector genomes ormore, and usually no more than 1×10¹⁵ vector genomes. In some cases, theamount of vector genomes that is delivered is at most about 1×10¹⁵vector genomes, e.g. 1×10¹⁴ vector genomes or less, for example 1×10¹³,1×10¹², 1×10¹¹, 1×10¹⁰, or 1×10⁹ vector genomes or less, in certaininstances 1×10⁸ vector genomes, and typically no less than 1×10⁸ vectorgenomes. In some cases, the amount of vector genomes that is deliveredis 1×10¹⁰ to 1×10¹¹ vector genomes. In some cases, the amount of vectorgenomes that is delivered is 1×10¹⁰ to 3×10¹² vector genomes. In somecases, the amount of vector genomes that is delivered is 1×10⁹ to 3×10¹³vector genomes. In some cases, the amount of vector genomes that isdelivered is 1×10⁸ to 3×10¹⁴ vector genomes.

In some cases, the amount of pharmaceutical composition to beadministered may be measured using multiplicity of infection (MOI). Insome cases, MOI may refer to the ratio, or multiple of vector or viralgenomes to the cells to which the nucleic may be delivered. In somecases, the MOI may be 1×10⁶. In some cases, the MOI may be 1×10⁵-1×10⁷.In some cases, the MOI may be 1×10⁴-1×10⁸. In some cases, recombinantviruses of the disclosure are at least about 1×10¹, 1×10², 1×10³, 1×10⁴,1×10⁵, 1×10⁶, 1×10⁷, 1×10⁸, 1×10⁹, 1×10¹⁰, 1×10¹¹, 1×10¹², 1×10¹³,1×10¹⁴, 1×10¹⁵, 1×10¹⁶, 1×10¹⁷, and 1×10¹⁸ MOI. In some cases,recombinant viruses of this disclosure are 1×10⁸ to 3×10¹⁴ MOI. In somecases, recombinant viruses of the disclosure are at most about 1×10¹,1×10², 1×10³, 1×10⁴, 1×10⁵, 1×10⁶, 1×10⁷, 1×10⁸, 1×10⁹, 1×10¹⁰, 1×10¹¹,1×10¹², 1×10¹³, 1×10¹⁴, 1×10¹⁵, 1×10¹⁶, 1×10¹⁷, and 1×10¹⁸ MOI. In someaspects, the amount of pharmaceutical composition comprises about 1×10⁸to about 1×10¹⁵ recombinant viruses, about 1×10⁹ to about 1×10¹⁴recombinant viruses, about 1×10¹⁰ to about 1×10¹³ recombinant viruses,about 1×10¹⁰ to about 3×10¹² recombinant viruses, or about 1×10¹¹ toabout 3×10¹² recombinant viruses.

Utility

Methods and compositions for the intravitreal delivery ofpolynucleotides to cone photoreceptors, and more particular fovealcones, find many uses in research and in medicine.

For example, such methods and compositions may be used in research totest the function of the gene product encode by the polynucleotide invivo, e.g. to better understand the function of the cone photoreceptorand/or whether the gene product will impact the viability and/orfunction of the cone photoreceptor.

As alluded to above, the subject rAAVs, referred to collectively hereinas “subject compositions”, find use in expressing a transgene in conecells of an animal, for example, in foveal cones of an animal. Forexample, the subject compositions may be used in research, e.g. todetermine the effect that the gene has on cone cell viability and/orfunction. As another example, the subject compositions may be used inmedicine, e.g. to treat a cone cell disorder. Thus, in some aspects ofthe invention, methods are provided for the expression of a gene in conecells, the method comprising contacting cone cells with a composition ofthe present disclosure. In some embodiments, contacting occurs in vitro.In some embodiments, contacting occurs in vivo, i.e., the subjectcomposition is administered to a subject.

For instances in which cone cells are to be contacted in vitro with asubject rAAV, the cells may be from any mammalian species, e.g. rodent(e.g. mice, rats, gerbils, squirrels), rabbit, feline, canine, goat,ovine, pig, equine, bovine, primate, human. Cells may be fromestablished cell lines, e.g. WERI cells, 661W cells, or they may beprimary cells, where “primary cells”, “primary cell lines”, and “primarycultures” are used interchangeably herein to refer to cells and cellscultures that have been derived from a subject and allowed to grow invitro for a limited number of passages, i.e. splittings, of the culture.For example, primary cultures are cultures that may have been passaged 0times, 1 time, 2 times, 4 times, 5 times, 10 times, or 15 times, but notenough times go through the crisis stage. Typically, the primary celllines of the present invention are maintained for fewer than 10 passagesin vitro.

If the cells are primary cells, they may be harvested from a mammal byany convenient method, e.g. whole explant, biopsy, etc. An appropriatesolution may be used for dispersion or suspension of the harvestedcells. Such solution will generally be a balanced salt solution, e.g.normal saline, PBS, Hank's balanced salt solution, etc., convenientlysupplemented with fetal calf serum or other naturally occurring factors,in conjunction with an acceptable buffer at low concentration, generallyfrom 5-25 mM. Convenient buffers include HEPES, phosphate buffers,lactate buffers, etc. The cells may be used immediately, or they may bestored, frozen, for long periods of time, being thawed and capable ofbeing reused. In such cases, the cells will usually be frozen in 10%DMSO, 50% serum, 40% buffered medium, or some other such solution as iscommonly used in the art to preserve cells at such freezingtemperatures, and thawed in a manner as commonly known in the art forthawing frozen cultured cells.

To promote expression of the transgene, the subject rAAV will becontacted with the cells for about 30 minutes to 24 hours or more, e.g.,1 hour, 1.5 hours, 2 hours, 2.5 hours, 3 hours, 3.5 hours 4 hours, 5hours, 6 hours, 7 hours, 8 hours, 12 hours, 16 hours, 18 hours, 20hours, 24 hours, etc. The subject rAAV may be provided to the subjectcells one or more times, e.g. one time, twice, three times, or more thanthree times, and the cells allowed to incubate with the agent(s) forsome amount of time following each contacting event e.g. 16-24 hours,after which time the media is replaced with fresh media and the cellsare cultured further. Contacting the cells may occur in any culturemedia and under any culture conditions that promote the survival of thecells. For example, cells may be suspended in any appropriate nutrientmedium that is convenient, such as Iscove's modified DMEM or RPMI 1640,supplemented with fetal calf serum or heat inactivated goat serum (about5-10%), L-glutamine, a thiol, particularly 2-mercaptoethanol, andantibiotics, e.g. penicillin and streptomycin. The culture may containgrowth factors to which the cells are responsive. Growth factors, asdefined herein, are molecules capable of promoting survival, growthand/or differentiation of cells, either in culture or in the intacttissue, through specific effects on a transmembrane receptor. Growthfactors include polypeptides and non-polypeptide factors.

Typically, an effective amount of subject rAAV is provided to producethe expression of the transgene in cells. As discussed elsewhere herein,the effective amount may be readily determined empirically, e.g. bydetecting the presence or levels of transgene gene product, by detectingan effect on the viability or function of the cone cells, etc.Typically, an effect amount of subject rAAV will promote greaterexpression of the transgene in cone cells than the same amount ofparental rAAV from which its capsid was derived. Typically, expressionwill be enhanced 2-fold or more relative to the expression from parentalrAAV, for example 3-fold, 4-fold, or 5-fold or more, in some instances10-fold, 20-fold or 50-fold or more, e.g. 100-fold.

In some embodiments, as when the transgene is a selectable marker, thepopulation of cells may be enriched for those comprising the transgeneby separating the modified cells from the remaining population.Separation may be by any convenient separation technique appropriate forthe selectable marker used. For example, if the transgene is afluorescent marker, cells may be separated by fluorescence activatedcell sorting, whereas if the transgene is a cell surface marker, cellsmay be separated from the heterogeneous population by affinityseparation techniques, e.g. magnetic separation, affinitychromatography, “panning” with an affinity reagent attached to a solidmatrix, or other convenient technique. Techniques providing accurateseparation include fluorescence activated cell sorters, which can havevarying degrees of sophistication, such as multiple color channels, lowangle and obtuse light scattering detecting channels, impedancechannels, etc. The cells may be selected against dead cells by employingdyes associated with dead cells (e.g. propidium iodide). Any techniquemay be employed which is not unduly detrimental to the viability of thecells. Cell compositions that are highly enriched for cells comprisingthe transgene are achieved in this manner. By “highly enriched”, it ismeant that the genetically modified cells will be 70% or more, 75% ormore, 80% or more, 85% or more, 90% or more of the cell composition, forexample, about 95% or more, or 98% or more of the cell composition. Inother words, the composition may be a substantially pure composition ofgenetically modified cells.

For instances in which cone cells are to be contacted in vivo with thesubject rAAV, the subject may be any mammal, e.g. rodent (e.g. mice,rats, gerbils), rabbit, feline, canine, goat, ovine, pig, equine,bovine, or primate. In certain embodiments, the subject is a primate ofthe Parvorder Catarrhini. As is known in the art, Catarrhini is one ofthe two subdivisions of the higher primates (the other being the NewWorld monkeys), and includes Old World monkeys and the apes, which inturn are further divided into the lesser apes or gibbons and the greatapes, consisting of the orangutans, gorillas, chimpanzees, bonobos, andhumans. In a further preferred embodiment, the primate is a human.

The subject rAAV may be administered to the retina of the subject by anysuitable method. For example, the subject composition may beadministered intraocularly via intravitreal injection or subretinalinjection. The general methods for delivering a vector via intravitrealinjection or via subretinal injection may be illustrated by thefollowing brief outlines. These examples are merely meant to illustratecertain features of the methods, and are in no way meant to be limiting.

For subretinal administration, the subject rAAV can be delivered in theform of a suspension injected subretinally under direct observationusing an operating microscope. Typically, a volume of 1 to 200 uL, e.g.50 uL, 100 uL, 150 ul, or 200 uL, but usually no more than 200 uL, ofthe subject composition will be administered by such methods. Thisprocedure may involve vitrectomy followed by injection of vectorsuspension using a fine cannula through one or more small retinotomiesinto the subretinal space. Briefly, an infusion cannula can be suturedin place to maintain a normal globe volume by infusion (of e.g. saline)throughout the operation. A vitrectomy is performed using a cannula ofappropriate bore size (for example 20 to 27 gauge), wherein the volumeof vitreous gel that is removed is replaced by infusion of saline orother isotonic solution from the infusion cannula. The vitrectomy isadvantageously performed because (1) the removal of its cortex (theposterior hyaloid membrane) facilitates penetration of the retina by thecannula; (2) its removal and replacement with fluid (e.g. saline)creates space to accommodate the intraocular injection of vector, and(3) its controlled removal reduces the possibility of retinal tears andunplanned retinal detachment.

For intravitreal administration, the subject rAAV can be delivered inthe form of a suspension. Initially, topical anesthetic is applied tothe surface of the eye followed by a topical antiseptic solution. Theeye is held open, with or without instrumentation, and the rAAV isinjected through the sclera with a short, narrow, for example a 30 gaugeneedle, into the vitreous cavity of the eye of a subject under directobservation. Typically, a volume of 1 to 100 uL, e.g. 25 uL, 50 uL, or100 uL, and usually no more than 100 uL, of the subject composition maybe delivered to the eye by intravitreal injection without removing thevitreous. Alternatively, a vitrectomy may be performed, and the entirevolume of vitreous gel is replaced by an infusion of the subjectcomposition. In such cases, up to about 4 mL of the subject compositionmay be delivered, e.g. to a human eye. Intravitreal administration isgenerally well tolerated. At the conclusion of the procedure, there issometimes mild redness at the injection site. There is occasionaltenderness, but most patients do not report any pain. No eye patch oreye shield is necessary after this procedure, and activities are notrestricted. Sometimes, an antibiotic eye drop is prescribed for severaldays to help prevent infection.

The subject methods and/or compositions may be used in medicine toexpress a therapeutic polynucleotide in cone photoreceptors as a therapyto treat or prevent a retinal disorder. The terms “treatment”,“treating” and the like are used herein to generally mean obtaining adesired pharmacologic and/or physiologic effect. The effect may beprophylactic in terms of completely or partially preventing a disease orsymptom thereof, e.g. reducing the likelihood that the disease orsymptom thereof occurs in the subject, and/or may be therapeutic interms of a partial or complete cure for a disease and/or adverse effectattributable to the disease. “Treatment” as used herein covers anytreatment of a disease in a mammal, and includes: (a) preventing thedisease from occurring in a subject which may be predisposed to thedisease but has not yet been diagnosed as having it; (b) inhibiting thedisease, i.e., arresting its development; or (c) relieving the disease,i.e., causing regression of the disease. The therapeutic agent may beadministered before, during or after the onset of disease or injury. Thetreatment of ongoing disease, where the treatment stabilizes or reducesthe undesirable clinical symptoms of the patient, is of particularinterest. Such treatment is desirably performed prior to complete lossof function in the affected tissues. The subject therapy will desirablybe administered during the symptomatic stage of the disease, and in somecases after the symptomatic stage of the disease.

There are a number of retinal disorders that may be treated or preventedusing the subject methods and/or compositions. Of particular interestare cone-associated disorders; that is, disorders that are associatedwith a loss of cone viability and/or a reduction in cone function. Asdiscussed above, cone photoreceptors are responsible for color visionand high acuity foveal vision, and are densely packed in a 1.5 mmdepression located in the center of the macula of the retina, called thefovea centralis. Consistent with this, disorders associated with conedysfunction and viability typically manifest in the macula and impactcolor vision and high acuity vision. Non-limiting examples ofcone-associated disorders include rod-cone dystrophy; cone-roddystrophy; progressive cone dystrophy; retinitis pigmentosa (RP);Stargardt Disease; macular telangiectasia, Leber hereditary opticneuropathy, Best's disease; adult vitelliform macular dystrophy;X-linked retinoschisis; color vision disorders such as blue conemonochromacy, achromatopsia, incomplete achromatopsia, protan defects,deutan defects, and tritan defects; and retinal disorders that affectthe central macula, such as, for example, age-related maculardegeneration, wet age-related macular degeneration, geographic atrophy,macular telangiectasia, retinitis pigmentosa, diabetic retinopathy,retinal vein occlusions, glaucoma, Sorsby's fundus dystrophy, adultvitelliform macular dystrophy, Best's disease, and X-linkedretinoschisis.

Stargardt's macular dystrophy. Stargardt's macular dystrophy, also knownas Stargardt Disease and fundus flavimaculatus, is an inherited form ofjuvenile macular degeneration that causes progressive vision lossusually to the point of legal blindness. The onset of symptoms usuallyappears between the ages of six and thirty years old (average of about16-18 years). Mutations in several genes, including ABCA4, CNGB3,ELOVL4, PROM1, are associated with the disorder. Symptoms typicallydevelop by twenty years of age, and include wavy vision, blind spots,blurriness, impaired color vision, and difficulty adapting to dimlighting. The main symptom of Stargardt disease is loss of visualacuity, which ranges from 20/50 to 20/200. In addition, those withStargardt disease are sensitive to glare; overcast days offer somerelief. Vision is most noticeably impaired when the macula is damaged,which can be observed by fundus exam.

Cone dystrophy. Cone dystrophy (COD) is an inherited ocular disordercharacterized by the loss of cone cells. The most common symptoms ofcone dystrophy are vision loss (age of onset ranging from the late teensto the sixties), sensitivity to bright lights, and poor color vision.Visual acuity usually deteriorates gradually, but it can deterioraterapidly to 20/200; later, in more severe cases, it drops to “countingfingers” vision. Color vision testing using color test plates (HRRseries) reveals many errors on both red-green and blue-yellow plates. Itis believed that the dystrophy is primary, since subjective andobjective abnormalities of cone function are found beforeophthalmoscopic changes can be seen. However, the retinal pigmentepithelium (RPE) rapidly becomes involved, leading to a retinaldystrophy primarily involving the macula. The fundus exam viaophthalmoscope is essentially normal early on in cone dystrophy, anddefinite macular changes usually occur well after visual loss. The mostcommon type of macular lesion seen during ophthalmoscopic examinationhas a bull's-eye appearance and consists of a doughnut-like zone ofatrophic pigment epithelium surrounding a central darker area. Inanother, less frequent form of cone dystrophy there is rather diffuseatrophy of the posterior pole with spotty pigment clumping in themacular area. Rarely, atrophy of the choriocapillaris and largerchoroidal vessels is seen in patients at an early stage. Fluoresceinangiography (FA) is a useful adjunct in the workup of someone suspectedto have cone dystrophy, as it may detect early changes in the retinathat are too subtle to be seen by ophthalmoscope. Because of the widespectrum of fundus changes and the difficulty in making the diagnosis inthe early stages, electroretinography (ERG) remains the best test formaking the diagnosis. Abnormal cone function on the ERG is indicated bya reduced single-flash and flicker response when the test is carried outin a well-lit room (photopic ERG). Mutations in several genes, includingGUCA1A, PDE6C, PDE6H, and RPGR, are associated with the disorder.

Cone-rod dystrophy. Cone-rod dystrophy (CRD, or CORD) is an inheritedretinal dystrophy that belongs to the group of pigmentary retinopathies.CRD is characterized by retinal pigment deposits visible on fundusexamination, predominantly localized to the macular region and the lossof both cone and rod cells. In contrast to rod-cone dystrophy (RCD)resulting from the primary loss in rod photoreceptors and later followedby the secondary loss in cone photoreceptors, CRD reflects the oppositesequence of events: primary cone involvement, or, sometimes, byconcomitant loss of both cones and rods. Symptoms include decreasedvisual acuity, color vision defects, photoaversion and decreasedsensitivity in the central visual field, later followed by progressiveloss in peripheral vision and night blindness. Mutations in severalgenes, including ADAM9, PCDH21, CRX, GUCY2D, PITPNM3, PROM1, PRPH2,RAX2, RIMS1, RPGR, and RPGRIP1, are associated with the disorder.

Spinocerebellar ataxia type 7. Spinocerebellar ataxia is a progressive,degenerative, inherited disease characterized by slowly progressiveincoordination of gait and is often associated with poor coordination ofhands, speech, and eye movements. There are multiple types of SCA, withSpinocerebellar ataxia type 7 (SCA-7) differing from most other SCAs inthat visual problems can occur in addition to poor coordination. SCA-7is associated with automosmal dominant mutations in the ATXN7/SCA7 gene.When the disease manifests itself before age 40, visual problems ratherthan poor coordination are typically the earliest signs of disease.Early symptoms include difficulty distinguishing colors and decreasedcentral vison. In addition, symptoms of ataxia (incoordination, slow eyemovements, and mild changes in sensation or reflexes) may be detectable.Loss of motor control, unclear speech, and difficulty swallowing becomeprominent as the disease progresses.

Bardet-Biedl syndrome-1. Bardet-Biedl syndrome-1 (BBS-1) is apleiotropic disorder with variable expressivity and a wide range ofclinical variability observed both within and between families. The mainclinical features are rod-cone dystrophy, with childhood-onset visualloss preceded by night blindness; postaxial polydactyly; truncal obesitythat manifests during infancy and remains problematic throughoutadulthood; specific learning difficulties in some but not allindividuals; male hypogenitalism and complex female genitourinarymalformations; and renal dysfunction, a major cause of morbidity andmortality. Vision loss is one of the major features of Bardet-Biedlsyndrome. Problems with night vision become apparent by mid-childhood,followed by blind spots that develop in the peripheral vision. Overtime, these blind spots enlarge and merge to produce tunnel vision. Mostpeople with Bardet-Biedl syndrome also develop blurred central vision(poor visual acuity) and become legally blind by adolescence or earlyadulthood. Bardet-Biedl syndrome can result from mutations in at least14 different genes (often called BBS genes) known or suspected to playcritical roles in cilia function, with mutations in BBS1 and BBS10 beingthe most common.

Achromatopsia. Achromatopsia, or Rod monochromatism, is a disorder inwhich subjects experience a complete lack of the perception of color,such that the subject sees only in black, white, and shades of grey.Other symptoms include reduced visual acuity, photophobia, nystagmus,small central scotoma, and eccentric fixation. The disorder isfrequently noticed first in children around six months of age by theirphotophobic activity and/or their nystagmus. Visual acuity and stabilityof the eye motions generally improve during the first 6-7 years of life(but remain near 20/200). Mutations in CNGB3, CNGA3, GNAT2, PDE6C, andPDE6HI have been associated with the disorder.

Incomplete achromatopsia. Incomplete achromatopsia is similar toAchromatopsia but with less penetrance. In incomplete achromatopsia, thesymptoms are similar to those of complete achromatopsia except in adiminished form. Individuals with incomplete achromatopsia have reducedvisual acuity with or without nystagmus or photophobia. Furthermore,these individuals show only partial impairment of cone cell function butagain have retained rod cell function.

Blue cone monochromacy. Blue cone (S cone) monochromatism (BCM) is arare X-linked congenital stationary cone dysfunction syndrome, affectingapproximately 1 in 100,000 individuals. Affected males with BCM have nofunctional long wavelength sensitive (L) or medium wavelength sensitive(M) cones in the retina, due to mutations at the genetic locus for the Land M-opsin genes. Color discrimination is severely impaired from birth,and vision is derived from the remaining preserved S cones and rodphotoreceptors. BCM typically presents with reduced visual acuity (6/24to 6/60), pendular nystagmus, photophobia, and patients often havemyopia. The rod-specific and maximal electroretinogram (ERG) usuallyshow no definite abnormality, whereas the 30 Hz cone ERG cannot bedetected. Single flash photopic ERG is often recordable, albeit smalland late, and the S cone ERG is well preserved.

Color vision deficiency. Color vision deficiency (CVD), or colorblindness, is the inability or decreased ability to see color, orperceive color differences, under normal lighting conditions.Individuals suffering from color blindness may be identified as suchusing any of a number of color vision tests, e.g., color ERG (cERG),pseudoisochromatic plates (Ishihara plates, Hardy-Rand-Ritterpolychromatic plates), the Farnsworth-Munsell 100 hue test, theFarnsworth's panel D-15, the City University test, Kollner's rule, etc.Examples of color vision deficiencies include protan defects, deutandefects, and tritan defects. Protan defects include protanopia (aninsensitivity to red light) and protanomaly (a reduced sensitivity tored light), and are associated with mutations in the L-Opsin gene(OPN1LW). Deutan defects include deuteranopia (an insensitivity to greenlight) and deutanomaly (a reduced sensitivity to green light), and areassociated with mutations in the M-Opsin gene (OPN1MW). Tritan defectsinclude tritanopia (an insensitivity to blue light) and tritanomaly (areduced sensitivity to blue light), and are associated with mutations inthe S-Opsin gene (OPN1SW).

Age-related macular degeneration. Age-related macular degeneration (AMD)is one of the leading causes of vision loss in people over the age of 50years. AMD mainly affects central vision, which is needed for detailedtasks such as reading, driving, and recognizing faces. The vision lossin this condition results from a gradual deterioration of photoreceptorsin the macula. Side (peripheral) vision and night vision are generallynot affected.

Researchers have described two major types of age-related maculardegeneration, known as the dry, or “nonexudative” form, and the wet, or“exudative” or “neovascular”, form, both of which may be treated bydelivering transgenes packaged in the subject rAAV.

Dry AMD is characterized by a buildup of yellow deposits called drusenbetween the retinal pigment epithelium and the underlying choroid of themacula, which may be observed by Fundus photography. This results in aslowly progressive loss of vision. The condition typically affectsvision in both eyes, although vision loss often occurs in one eye beforethe other. Other changes may include pigment changes and RPE atrophy.For example, in certain cases called central geographic atrophy, or“GA”, atrophy of the retinal pigment epithelial and subsequent loss ofphotoreceptors in the central part of the eye is observed. Dry AMD hasbeen associated with mutations in CD59 and genes in the complementcascade.

Wet AMD is a progressed state of dry AMD, and occurs in abut 10% of dryAMD patients. Pathological changes include retinal pigment epithelialcells (RPE) dysfunction, fluid collecting under the RPE, and choroidalneovascularization (CNV) in the macular area. Fluid leakage, RPE orneural retinal detachment and bleeding from ruptured blood vessels canoccur in severe cases. Symptoms of wet AMD may include visualdistortions, such as straight lines appearing wavy or crooked, a doorwayor street sign looking lopsided, or objects appearing smaller or fartheraway than they really are; decreased central vision; decreased intensityor brightness of colors; and well-defined blurry spot or blind spot inthe field of vision. Onset may be abrupt and worsen rapidly. Diagnosismay include the use of an Amsler grid to test for defects in thesubject's central vision (macular degeneration may cause the straightlines in the grid to appear faded, broken or distorted), fluoresceinangiogram to observe blood vessel or retinal abnormalities, and opticalcoherence tomography to detect retina swelling or leaking blood vessels.A number of cellular factors have been implicated in the generation ofCNV, among which are vascular endothelial growth factor (VEGF),platelet-derived growth factor (PDGF), pigment epithelium-derived factor(PEDF), hypoxia inducible factor (HIF), angiopoietin (Ang), and othercytokines, mitogen-activated protein kinases (MAPK) and others.

Macular telangiectasia. Macular telangiectasia (MacTel) is a form ofpathologically dilated blood vessels (telangiectasia) in the parafovealregion of the macula. The tissue deteriorates and the retinal structurebecomes scarred due to the development of liquid-filled cysts, whichimpairs nutrition of the photoreceptor cells and destroys visionpermanently. There are two types of MacTel, type 1 and type 2. Maculartelangiectasia type 2 is a bilateral disease, whose prevalence hasrecently been shown to be as high as 0.1% in persons 40 years and older.Biomicroscopy may show reduced retinal transparency, crystallinedeposits, mildly ectatic capillaries, blunted venules, retinal pigmentplaques, foveal atrophy, and neovascular complexes. Fluoresceinangiography shows telangiectatic capillaries predominantly temporal tothe foveola in the early phase and a diffuse hyperfluorescence in thelate phase. High-resolution optical coherence tomography (OCT) mayreveal disruption of the photoreceptor inner segment-outer segmentborder, hyporeflective cavities at the level of the inner or outerretina, and atrophy of the retina in later stages. In Type 1 maculartelangiectasia, the disease almost always occurs in one eye, whichdifferentiates it from Type 2. While MacTel does not usually cause totalblindness, it commonly causes loss of the central vision, which isrequired for reading and driving vision, over a period of 10-20 years.

Retinitis pigmentosa. Retinitis Pigmentosa (RP) is a group of inheriteddisorders characterized by progressive peripheral vision loss and nightvision difficulties (nyctalopia) that can lead to central vision loss.Presenting signs and symptoms of RP vary, but the classic ones includenyctalopia (night blindness, most commonly the earliest symptom in RP);visual loss (usually peripheral, but in advanced cases, central visualloss); and photopsia (seeing flashes of light). Because RP is acollection of many inherited diseases, significant variability exists inthe physical findings. Ocular examination involves assessment of visualacuity and pupillary reaction, as well as anterior segment, retinal, andfunduscopic evaluation. In some instances, the RP is one aspect of asyndrome, e.g. syndromes that are also associated with hearing loss(Usher syndrome, Waardenburg syndrome, Alport syndrome, Refsum disease);Kearns-Sayre syndrome (external ophthalmoplegia, lid ptosis, heartblock, and pigmentary retinopathy); Abetalipoproteinemia (Fatmalabsorption, fat-soluble vitamin deficiencies, spinocerebellardegeneration, and pigmentary retinal degeneration);mucopolysaccharidoses (eg, Hurler syndrome, Scheie syndrome, Sanfilipposyndrome); Bardet-Biedl syndrome (Polydactyly, truncal obesity, kidneydysfunction, short stature, and pigmentary retinopathy); and neuronalceroid lipofuscinosis (Dementia, seizures, and pigmentary retinopathy;infantile form is known as Jansky-Bielschowsky disease, juvenile form isVogt-Spielmeyer-Batten disease, and adult form is Kufs syndrome).Retinitis pigmentosa is most commonly associated with mutations in theRHO, RP2, RPGR, RPGRIP1, PDE6A, PDE6B, MERTK, PRPH2, CNGB1, USH2A,ABCA4, BBS genes.

Diabetic retinopathy. Diabetic retinopathy (DR) is damage to the retinacaused by complications of diabetes, which can eventually lead toblindness. Without wishing to be bound by theory, it is believed thathyperglycemia-induced intramural pericyte death and thickening of thebasement membrane lead to incompetence of the vascular walls. Thesedamages change the formation of the blood-retinal barrier and also makethe retinal blood vessels become more permeable.

There are two stages of diabetic retinopathy: non-proliferative diabeticretinopathy (NPDR), and proliferative diabetic retinopathy (PDR).Nonproliferative diabetic retinopathy is the first stage of diabeticretinopathy, and is diagnosed by fundoscopic exam and coexistentdiabetes. In cases of reduced vision, fluorescein angiography may bedone to visualize the vessles in the back of the eye to and any retinalischemia that may be present. All people with diabetes are at risk fordeveloping NPDR, and as such, would be candidates for prophylactictreatment with the subject vectors. Proliferative diabetic retinopathyis the second stage of diabetic retinopathy, characterized byneovascularization of the retina, vitreous hemorrhage, and blurredvision. In some instances, fibrovascular proliferation causes tractionalretinal detachment. In some instances, the vessels can also grow intothe angle of the anterior chamber of the eye and cause neovascularglaucoma. Individuals with NPDR are at increased risk for developingPDR, and as such, would be candidates for prophylactic treatment withthe subject vectors.

Diabetic macular edema. Diabetic macular edema (DME) is an advanced,vision-limiting complication of diabetic retinopathy that affects nearly30% of patients who have had diabetes for at least 20 years, and isresponsible for much of the vision loss due to DR. It results fromretinal microvascular changes that compromise the blood-retinal barrier,causing leakage of plasma constituents into the surrounding retina and,consequently, retinal edema. Without wishing to be bound by theory, itis believed that hyperglycemia, sustained alterations in cell signalingpathways, and chronic microvascular inflammation with leukocyte-mediatedinjury leads to chronic retinal microvascular damage, which triggers anincrease in intraocular levels of VEGF, which in turn increases thepermeability of the vasculature.

Patients at risk for developing DME include those who have had diabetesfor an extended amount of time and who experience one or more of severehypertension (high blood pressure), fluid retention, hypoalbuminemia, orhyperlipidemia. Common symptoms of DME are blurry vision, floaters,double vision, and eventually blindness if the condition is allowed toprogress untreated. DME is diagnosed by funduscopic examination asretinal thickening within 2 disc diameters of the center of the macula.Other methods that may be employed include Optical coherence tomography(OCT) to detect retinal swelling, cystoid edema, and serous retinaldetachment; fluorescein angiography, which distinguishes and localizesareas of focal versus diffuse leakage, thereby guiding the placement oflaser photocoagulation if laser photocoagulation is to be used to treatthe edema; and color stereo fundus photographs, which can be used toevaluate long-term changes in the retina. Visual acuity may also bemeasured, especially to follow the progression of macular edema andobserve its treatment following administration of the subjectpharmaceutical compositions.

Retinal vein occlusions. A retinal vein occlusion (RVO) is a blockage ofthe portion of the circulation that drains the retina of blood. Theblockage can cause back-up pressure in the capillaries, which can leadto hemorrhages and also to leakage of fluid and other constituents ofblood.

Glaucoma. Glaucoma is a term describing a group of ocular (eye)disorders that result in optic nerve damage, often associated withincreased fluid pressure in the eye (intraocular pressure)(IOP). Thedisorders can be roughly divided into two main categories, “open-angle”and “closed-angle” (or “angle closure”) glaucoma. Open-angle glaucomaaccounts for 90% of glaucoma cases in the United States. It is painlessand does not have acute attacks. The only signs are graduallyprogressive visual field loss, and optic nerve changes (increasedcup-to-disc ratio on fundoscopic examination). Closed-angle glaucomaaccounts for less than 10% of glaucoma cases in the United States, butas many as half of glaucoma cases in other nations (particularly Asiancountries). About 10% of patients with closed angles present with acuteangle closure crises characterized by sudden ocular pain, seeing halosaround lights, red eye, very high intraocular pressure (>30 mmHg),nausea and vomiting, suddenly decreased vision, and a fixed, mid-dilatedpupil. It is also associated with an oval pupil in some cases.Modulating the activity of proteins encoded by DLK, NMDA, INOS, CASP-3,Bcl-2, or Bcl-xl may treat the condition.

Sorsby's fundus dystrophy. Sorsby's fundus dystrophy is an autosomaldominant, retinal disease associated with mutations in the TIMP3 gene.Clinically, early, mid-peripheral, drusen and colour vision deficits arefound. Some patients complain of night blindness. Most commonly, thepresenting symptom is sudden acuity loss, manifest in the third tofourth decades of life, due to untreatable submacularneovascularisation. Histologically, there is accumulation of a confluentlipid containing material 30 μm thick at the level of Bruch's membrane.

Vitelliform macular dystrophy. Vitelliform macular dystrophy is agenetic eye disorder that can cause progressive vision loss. Vitelliformmacular dystrophy is associated with the buildup of fatty yellow pigment(lipofuscin) in cells underlying the macula. Over time, the abnormalaccumulation of this substance can damage cells that are critical forclear central vision. As a result, people with this disorder often losetheir central vision, and their eyesight may become blurry or distorted.Vitelliform macular dystrophy typically does not affect side(peripheral) vision or the ability to see at night.

Researchers have described two forms of vitelliform macular dystrophywith similar features. The early-onset form (known as Best disease)usually appears in childhood; the onset of symptoms and the severity ofvision loss vary widely. It is associated with mutations in theVMD2/BEST1 gene. The adult-onset form (Adult vitelliform maculardystrophy) begins later, usually in mid-adulthood, and tends to causevision loss that worsens slowly over time. It has been associated withmutations in the PRPH2 gene. The two forms of vitelliform maculardystrophy each have characteristic changes in the macula that can bedetected during an eye examination.

Rod-cone dystrophy. Rod-cone dystrophies are a family of progressivediseases in which rod dysfunction, which leads to night blindness andloss of peripheral visual field expanses, is either the prevailingproblem or occurring at least as severely as cone dysfunction. Ascallop-bordered lacunar atrophy may be seen in the midperiphery of theretina. The macula is only mildly involved by clinical examinationalthough central retinal thinning is seen in all cases. Dyschromatopsiais mild early and usually becomes more severe. The visual fields aremoderately to severely constricted although in younger individuals atypical ring scotoma is present. The peripheral retina contains ‘whitedots’ and often resembles the retinal changes seen in retinitis punctatealbescens. Retinitis pigmentosa is the main group of diseases includedunder this definition and, as a whole, is estimated to affectapproximately one in every 3,500 people. Depending on the classificationcriteria used, about 60-80% of all retinitis pigmentosa patients have aclear-cut rod-cone dystrophy pattern of retinal disease and once othersyndromic forms are taken into account, about 50-60% of all retinitispigmentosas fall in the rod-cone dystrophy nonsyndromic category.

Leber's congenital amaurosis. Leber's congenital amaurosis (LCA) is asevere dystrophy of the retina that typically becomes evident in thefirst year of life. Visual function is usually poor and oftenaccompanied by nystagmus, sluggish or near-absent pupillary responses,photophobia, high hyperopia, and keratoconus. Visual acuity is rarelybetter than 20/400. A characteristic finding is Franceschetti'soculo-digital sign, comprising eye poking, pressing, and rubbing. Theappearance of the fundus is extremely variable. While the retina mayinitially appear normal, a pigmentary retinopathy reminiscent ofretinitis pigmentosa is frequently observed later in childhood. Theelectroretinogram (ERG) is characteristically “nondetectable” orseverely subnormal. Mutations in 17 genes are known to cause LCA: GUCY2D(locus name: LCA1), RPE65 (LCA2), SPATA7 (LCA3), AIPL1 (LCA4), LCAS(LCAS), RPGRIP1 (LCA6), CRX (LCA7), CRB1 (LCAS), NMNAT1 (LCA9), CEP290(LCA10), IMPDH1 (LCA11), RD3 (LCA12), RDH12 (LCA13), LRAT (LCA14), TULP1(LCA15), KCNJ13 (LCA16), and IQCB1. Together, mutations in these genesare estimated to account for over half of all LCA diagnoses. At leastone other disease locus for LCA has been reported, but the gene is notknown.

X-linked retinoschisis. X-linked retinoschisis (XLRS) is characterizedby symmetric bilateral macular involvement with onset in the firstdecade of life, in some cases as early as age three months. Fundusexamination shows areas of schisis (splitting of the nerve fiber layerof the retina) in the macula, sometimes giving the impression of a spokewheel pattern. Schisis of the peripheral retina, predominantlyinferotemporally, occurs in approximately 50% of individuals. Affectedmales typically have vision of 20/60 to 20/120. Visual acuity oftendeteriorates during the first and second decades of life but thenremains relatively stable until the fifth or sixth decade. The diagnosisof X-linked juvenile retinoschisis is based on fundus findings, resultsof electrophysiologic testing, and molecular genetic testing. RS1 is theonly gene known to be associated with X-linked juvenile retinoschisis.

An individual affected by a cone cell disorder or at risk for developinga cone cell disorder can be readily identified using techniques todetect the symptoms of the disorder as known in the art, including,without limitation, fundus photography; Optical coherence tomography(OCT); adaptive optics (AO); electroretinography, e.g. ERG, color ERG(cERG); color vision tests such as pseudoisochromatic plates (Ishiharaplates, Hardy-Rand-Ritter polychromatic plates), the Farnsworth-Munsell100 hue test, the Farnsworth's panel D-15, the City university test,Kollner's rule, and the like; and visual acuity tests such as the ETDRSletters test, Snellen visual acuity test, visual field test, contrastsensitivity test, and the like; as will be known by the ordinarilyskilled artisan. Additionally or alternatively, the individual affectedby a cone cell disorder or at risk for developing a cone cell disordercan be readily identified using techniques to detect gene mutations thatare associated with the cone cell disorder as known in the art,including, without limitation, PCR, DNA sequence analysis, restrictiondigestion, Southern blot hybridization, mass spectrometry, etc. In someembodiments, the method comprises the step of identifying the individualin need of a cone cell therapy. In such instances, any convenient methodfor determining if the individual has the symptom(s) of a cone celldisorder or is at risk for developing a cone cell disorder, for exampleby detecting the symptoms described herein or known in the art, bydetecting a mutation in a gene as herein or as known in the art, etc.may be utilized to identify the individual in need of a cone celltherapy.

In practicing the subject methods, the subject composition is typicallydelivered to the retina of the subject in an amount that is effective toresult in the expression of the transgene in the cone cells. In someembodiments, the method comprises the step of detecting the expressionof the transgene in the cone cells.

There are a number of ways to detect the expression of a transgene, anyof which may be used in the subject embodiments. For example, expressionmay be detected directly, i.e. by measuring the amount of gene product,for example, at the RNA level, e.g. by RT-PCR, Northern blot, RNAseprotection; or at the protein level, e.g. by Western blot, ELISA,immunohistochemistry, and the like. As another example, expression maybe detected indirectly, i.e. by detecting the impact of the gene producton the viability or function of the cone photoreceptor in the subject.For example, if the gene product encoded by the transgene improves theviability of the cone cell, the expression of the transgene may bedetected by detecting an improvement in viability of the cone cell, e.g.by fundus photography, Optical coherence tomography (OCT), AdaptiveOptics (AO), and the like. If the gene product encoded by the transgenealters the activity of the cone cell, the expression of the transgenemay be detected by detecting a change in the activity of the cone cell,e.g. by electroretinogram (ERG) and color ERG (cERG); functionaladaptive optics; color vision tests such as pseudoisochromatic plates(Ishihara plates, Hardy-Rand-Ritter polychromatic plates), theFarnsworth-Munsell 100 hue test, the Farnsworth's panel D-15, the Cityuniversity test, Kollner's rule, and the like; and visual acuity testssuch as the ETDRS letters test, Snellen visual acuity test, visual fieldtest, contrast sensitivity test, and the like, as a way of detecting thepresence of the delivered polynucleotide. In some instances, both animprovement in viability and a modification in cone cell function may bedetected.

In some embodiments, the subject method results in a therapeuticbenefit, e.g. preventing the development of a disorder, halting theprogression of a disorder, reversing the progression of a disorder, etc.In some embodiments, the subject method comprises the step of detectingthat a therapeutic benefit has been achieved. The ordinarily skilledartisan will appreciate that such measures of therapeutic efficacy willbe applicable to the particular disease being modified, and willrecognize the appropriate detection methods to use to measuretherapeutic efficacy. For example, therapeutic efficacy in treatingmacular degeneration may be observed as a reduction in the rate ofmacular degeneration or a cessation of the progression of maculardegeneration, effects which may be observed by, e.g., fundusphotography, OCT, or AO, by comparing test results after administrationof the subject composition to test results before administration of thesubject composition. As another example, therapeutic efficacy intreating a progressive cone dysfunction may be observed as a reductionin the rate of progression of cone dysfunction, as a cessation in theprogression of cone dysfunction, or as an improvement in cone function,effects which may be observed by, e.g., ERG and/or cERG; color visiontests; functional adaptive optics; and/or visual acuity tests, forexample, by comparing test results after administration of the subjectcomposition to test results before administration of the subjectcomposition and detecting a change in cone viability and/or function. Asa third example, therapeutic efficacy in treating a color visiondeficiency may be observed as an alteration in the individual'sperception of color, e.g. in the perception of red wavelengths, in theperception of green wavelengths, in the perception of blue wavelengths,effects which may be observed by, e.g., cERG and color vision tests, forexample, by comparing test results after administration of the subjectcomposition to test results before administration of the subjectcomposition and detecting a change in cone viability and/or function.

Expression of a transgene delivered by the subject rAAV is expected tobe robust. Accordingly, in some instances, the expression of thetransgene, e.g. as detected by measuring levels of gene product, bymeasuring therapeutic efficacy, etc, may be observed two months or lessafter administration, e.g. 4, 3 or 2 weeks or less after administration,for example, 1 week after administration of the subject composition.Expression of the transgene is also expected to persist over time.Accordingly, in some instances, the expression of the transgene, e.g. asdetected by measuring levels of gene product, by measuring therapeuticefficacy, etc., may be observed 2 months or more after administration ofthe subject composition, e.g., 4, 6, 8, or 10 months or more, in someinstances 1 year or more, for example 2, 3, 4, or 5 years, in certaininstances, more than 5 years.

In certain embodiments, the method comprises the step of detectingexpression of the polynucleotide delivered by the subject rAAV in thecone cells, wherein expression is enhanced relative to expression froman AAV not comprising a 7-10 amino acid insert in the GH loop. i.e. areference control, e.g. a parental rAAV into which the peptide has beeninserted. Typically, expression will be enhanced 2-fold or more relativeto the expression from a reference, e.g. a parental rAAV, for example3-fold, 4-fold, or 5-fold or more, in some instances 10-fold, 20-fold or50-fold or more, e.g. 100-fold, as evidenced by, e.g. earlier detection,higher levels of gene product, a stronger functional impact on thecells, etc.

Typically, an effective amount to achieve a change in will be about1×10⁸ vector genomes or more, in some cases 1×10⁹, 1×10¹⁰, 1×10¹¹,1×10¹², or 1×10¹³ vector genomes or more, in certain instances, 1×10¹⁴vector genomes or more, and usually no more than 1×10¹⁵ vector genomes.In some cases, the amount of vector genomes that is delivered is at mostabout 1×10¹⁵ vector genomes, e.g. 1×10¹⁴ vector genomes or less, forexample 1×10¹³, 1×10¹², 1×10¹¹, 1×10¹⁰, or 1×10⁹ vector genomes or less,in certain instances 1×10⁸ vector genomes, and typically no less than1×10⁸ vector genomes. In some cases, the amount of vector genomes thatis delivered is 1×10¹⁰ to 1×10¹¹ vector genomes. In some cases, theamount of vector genomes that is delivered is 1×10¹⁰ to 3×10¹² vectorgenomes. In some cases, the amount of vector genomes that is deliveredis 1×10⁹ to 3×10¹³ vector genomes. In some cases, the amount of vectorgenomes that is delivered is 1×10⁸ to 3×10¹⁴ vector genomes.

In some cases, the amount of pharmaceutical composition to beadministered may be measured using multiplicity of infection (MOI). Insome cases, MOI may refer to the ratio, or multiple of vector or viralgenomes to the cells to which the nucleic may be delivered. In somecases, the MOI may be 1×10⁶. In some cases, the MOI may be 1×10⁵-1×10⁷.In some cases, the MOI may be 1×10⁴-1×10⁸. In some cases, recombinantviruses of the disclosure are at least about 1×10¹, 1×10², 1×10³, 1×10⁴,1×10⁵, 1×10⁶, 1×10⁷, 1×10⁸, 1×10⁹, 1×10¹⁰, 1×10¹¹, 1×10¹², 1×10¹³,1×10¹⁴, 1×10¹⁵, 1×10¹⁶, 1×10¹⁷, and 1×10¹⁸ MOI. In some cases,recombinant viruses of this disclosure are 1×10⁸ to 3×10¹⁴ MOI. In somecases, recombinant viruses of the disclosure are at most about 1×10¹,1×10², 1×10³, 1×10⁴, 1×10⁵, 1×10⁶, 1×10⁷, 1×10⁸, 1×10⁹, 1×10¹⁰, 1×10¹¹,1×10¹², 1×10¹³, 1×10¹⁴, 1×10¹⁵, 1×10¹⁶, 1×10¹⁷, and 1×10¹⁸ MOI. In someaspects, the amount of pharmaceutical composition comprises about 1×10⁸to about 1×10¹⁵ particles of recombinant viruses, about 1×10⁹ to about1×10¹⁴ particles of recombinant viruses, about 1×10¹⁰ to about 1×10¹³particles of recombinant viruses, or about 1×10¹¹ to about 3×10¹²particles of recombinant viruses.

Individual doses are typically not less than an amount required toproduce a measurable effect on the subject, and may be determined basedon the pharmacokinetics and pharmacology for absorption, distribution,metabolism, and excretion (“ADME”) of the subject composition or itsby-products, and thus based on the disposition of the composition withinthe subject. This includes consideration of the route of administrationas well as dosage amount, which can be adjusted for subretinal (applieddirectly to where action is desired for mainly a local effect),intravitreal (applied to the vitreaous for a pan-retinal effect), orparenteral (applied by systemic routes, e.g. intravenous, intramuscular,etc.) applications. Effective amounts of dose and/or dose regimen canreadily be determined empirically from preclinical assays, from safetyand escalation and dose range trials, individual clinician-patientrelationships, as well as in vitro and in vivo assays such as thosedescribed herein and illustrated in the Experimental section, below.

All of the above U.S. patents, U.S. patent application publications,U.S. patent applications, foreign patents, foreign patent applicationsand non-patent publications referred to in this specification and/orlisted in the Application Data Sheet, are incorporated herein byreference, in their entirety.

From the foregoing it will be appreciated that, although specificembodiments of the invention have been described herein for purposes ofillustration, various modifications may be made without deviating fromthe spirit and scope of the invention. Accordingly, the invention is notlimited except as by the appended claims.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the present invention, and are not intended to limit thescope of what the inventors regard as their invention nor are theyintended to represent that the experiments below are all or the onlyexperiments performed. Efforts have been made to ensure accuracy withrespect to numbers used (e.g. amounts, temperature, etc.) but someexperimental errors and deviations should be accounted for. Unlessindicated otherwise, parts are parts by weight, molecular weight isweight average molecular weight, temperature is in degrees Centigrade,and pressure is at or near atmospheric.

General methods in molecular and cellular biochemistry can be found insuch standard textbooks as Molecular Cloning: A Laboratory Manual, 3rdEd. (Sambrook et al., HaRBor Laboratory Press 2001); Short Protocols inMolecular Biology, 4th Ed. (Ausubel et al. eds., John Wiley & Sons1999); Protein Methods (Bollag et al., John Wiley & Sons 1996); NonviralVectors for Gene Therapy (Wagner et al. eds., Academic Press 1999);Viral Vectors (Kaplift & Loewy eds., Academic Press 1995); ImmunologyMethods Manual (I. Lefkovits ed., Academic Press 1997); and Cell andTissue Culture: Laboratory Procedures in Biotechnology (Doyle &Griffiths, John Wiley & Sons 1998), the disclosures of which areincorporated herein by reference. Reagents, cloning vectors, and kitsfor genetic manipulation referred to in this disclosure are availablefrom commercial vendors such as BioRad, Stratagene, Invitrogen,Sigma-Aldrich, and ClonTech.

Example 1 Background

New therapies are needed for the treatment of many cone photoreceptorassociated disorders, including macular dystrophies such as cone-roddystrophy, cone dystrophy, Stargardt macular dystrophy, andachromatopsia; color vision disorders such as protan, deutan, and tritandefects; and vision disorders of the central macula such as age-relatedmacular degeneration, macular telangiectasia, retinitis pigmentosa,diabetic retinopathy, retinal vein occlusions, glaucoma, Sorsby's fundusdystrophy, adult vitelliform macular dystrophy, Best's disease, andX-linked retinoschisis. As these vision disorders are associated with aloss of function and/or viability of the cone photoreceptors, it ishypothesized that these disorders may be treatable by delivering atherapeutic gene to cone photoreceptors to rescue cone viability andfunction.

To that end, the polynucleotide cassette “pMNTC” was designed in whichenhancer, promoter, 5′UTR, intron, Kozak, and polyadenylation sequenceswere designed for cone-specific expression (FIG. 6a ). The cassetteincluded an LCR enhancer sequence from the L- and M-opsin genomic locusand a truncated promoter sequence from the M-Opsin gene, comprisingabout 140 nucleotides upstream of the transcriptional start site. Inaddition, the cassette included a 5′ untranslated region (5′ UTR) basedon the M-opsin 5′UTR but modified to have minimal secondary structureand to include additional sequence at its 3′ end into which an intronwas inserted. The intronic sequence used was a pSI chimeric intronhaving the 5′-donor site from the first intron of the human β-globingene and the branch and 3′-acceptor site from the intron that liesbetween the leader and the body of an immunoglobulin gene heavy chainvariable region (Bothwell, A. L. et al. (1981) Heavy chain variableregion contribution to the NPb family of antibodies: Somatic mutationevident in a gamma 2a variable region. Cell 24, 625-37). The sequencesof the donor and acceptor sites, along with the branchpoint site, werechanged to match the consensus sequences for splicing (Senapathy, P.,Shapiro, M. B. and Harris, N. L. (1990) Meth. Enzymol. 183, 252-78).Also included in the pMNTC polynucleotide cassette was a strong Kozaksequence and an SV40 polyadenylation sequence.

Experiments were also performed to identify the best AAV with which todeliver transgenes to cone cells. Successful delivery of polynucleotidesto cells of the retina for the purposes of gene therapy has beenachieved using viral vectors such as AAV and lentivirus. However, theseviruses must be injected subretinally to reach the cells of thenon-human primate (NHP) retina, a procedure that carries with it therisk of retinal damage. A less disruptive approach is administration byintravitreal injection. However, efficient transduction of conephotoreceptors following intravitreal delivery of AAV or lentivirus hasnever been demonstrated: while reports exist of AAVs with the ability totransduce retinal cone cells with high efficiency (Merigan et al. IOVS2008, 49 E-abstract 4514), later reports have questioned the efficacy ofthese vectors (Yin et al. IOVS 2011, 52(5):2775-2783).

Results

Directed evolution of AAV2 has led to the identification of the viralvariant “7m8” that is able to transduce photoreceptors better than wildtype AAV2 (Dalkara et al. Sci Transl Med 2013). However, the retinacontains two types of photoreceptors—rods and cones—and no reports existdemonstrated whether AAV2-7m8 can transduce cone photoreceptors, per se,and more particularly, cone photoreceptors in the highly cone-enrichedarea of the fovea. To test this possibility, we delivered AAV2-7m8carrying an expression cassette of the ubiquitous promoter CMV operablylinked to GFP to the retina of African Green monkey by intravitrealinjection. Intravitreally delivered AAV2-7m8.CMV.GFP appeared totransduce retinal cells in the fovea centralis (the 0.35 mm diameterrod-free region of retina at the center of the foveal pit) and parafovea(the lip of the depression) of primates more efficiently thanintravitreally-delivered AAV2 or other AAV variants previously shown inthe art to transduce retinal cells. Neither AAV2-7m8 nor the other AAVstested appeared to be able to transduce the cones of the primate fovea,the 1.5 mm-diameter cone-enriched region of retina that surrounds thefoveola and forms the slopes of the pit (FIG. 1).

We next packaged a genome comprising pMNTC operably linked to GFP withinthe AAV2-7m8 capsid, and assessed the ability of this vector compositionto express the GFP transgene in cone cells in vivo when injectedintravitreally. Expression was evaluated in a number of species withvarying numbers of retinal cones cells among total photoreceptors,including mouse (3% cones), rat (1% cones), gerbil (13% cones), andnonhuman primate (5% cones). Contrary to our results in FIG. 1, stronggene expression could be detected throughout the nonhuman primate fovea(FIG. 2). These data indicate that intravitreally delivered AAV2-7m8can, in fact, transduce retinal cones, and that pMNTC acts as a robustexpression cassette in cone cells. Robust reporter gene expression wasalso seen in the intravireally injected retina of the rat (data notshown) and gerbil (FIG. 4A), with expression levels and anatomiclocation correlating with cone abundance and location in all species.

To determine the cell-specificity of pMNTC-directed expression, wholemounts of transduced mouse retina were analyzed by immunohistochemistryusing an antibody that is specific for cone L and M opsins. Theexpression of L/M opsin, which labels the outer segments of conephotoreceptors only, was observed in virtually all of the cones of themouse retina that expressed GFP from the AAV2-7m8.MNTC.GFP vector (FIG.3), indicating that MNTC-directed expression of transgenes is highlycone-specific. Moreover 80% or more of the cone outer segments that werelabelled by the L/M opsin-specific antibody also expressed the GFPtransgene, indicating that AAV2-7m8 transduces cones highly efficiently(FIG. 3).

We also determined the cell-specificity of pR2.1-directed expression bypackaging a genome comprising pR2.1 operably linked to GFP within theAAV2-7m8 capsid (AAV2-7m8pR2.1.GFP vector). pR2.1 comprises the humanL/M opsin enhancer (“LCR”) and the promoter region from the humanL-Opsin gene. In addition, pR2.1 comprises the L-Opsin 5′UTR fused toadditional 5′UTR sequence at its 3′ end, into which modified SV40 late16s intronic sequence has been inserted. This is followed by the L-OpsinKozak sequence, which is then typically linked in-frame to a transgene.At the end of the cassette is an SV40 polyA tail. The ability of thisvector composition to express the GFP transgene in cone cells in vivowas assessed 12 weeks after intravitreal injection in an African greenmonkey (non-human primate; NHP). Briefly, the NHP received bilateralintravitreal administrations of 50 uL of 1.0×10¹³ vg/mLAAV2-7m8pR2.1.GFP to yield a final dose of 5×10¹¹ vg per eye. Retinalexamination, including fundus color and fluorescence photography, wasperformed by using a Topcon TRC-50EX retinal camera with Canon 6Ddigital imaging hardware and a Spectralis OCT Plus at baseline and atweeks 4, 8, and 12 post-intravitreal vector injection. The animal wasterminated at 12 weeks and eyes processed. A cross-section of a treatedretina from the NHP was stained with a chicken polyclonal anti-GFPantibody (Abcam Cat #13970; Cambridge, UK); a rabbit polyclonal anti-L/MOpsin antibody specific for opsin cones (Abcam Cat #5405); a 1D4 mousemonoclonal anti-rhodopsin antibody (Abcam Cat #5417); and Dapi to stainall nuclei (Invitrogen Ref #D21490). GFP-tagged transgene containingcells were imaged by multispectral analysis along with the antibodyprobes and DIC (differential interference contract for topology). Asflattened stacks of optical planes through the entire section. Cellanalysis for transgene was optimized using morphology and colocalizationwith probes. GFP (transgene) staining co-localized with L/M opsinstaining and not with rhodopsin staining, indicating that pR2.1 promotesexpression in cone cells specifically (FIG. 7). GFP transgene signal wasobserved at fovea, mid and far periphery; GFP transgene signalcolocalized with L/M-opsin, calbindin and PNA probe; clear exclusion of1D4-containing cells in fovea was observed; and there was no GFPtransgene positive cell association with rods or other probe-containingcells. (FIG. 7). In summary, cells double-stained for GFP (transgeneexpression) and L/M opsin were observed, but there was a lack of cellsdouble-staining for both GFP and rhodopsin, indicating that theAAV2-7m8pR2.1.GFP vector specifically directed expression in cone cellsand not rod cells.

We next compared the ability of pMNTC to promote expression in conecells to that of pR2.1. Viral preparations of AAV2-7m8.MNTC.GFP andAAV2-7m8.pR2.1.GFP were delivered intravitreally to the retinas ofgerbils and nonhuman primates in vivo, and the retinas imaged in vivo 2weeks, 4 weeks, 8 weeks, and 12 weeks later by fundus autofluorescenceand OCT. GFP reporter expression was detected sooner, more strongly, andin more cones in gerbil retina transduced with rAAV carrying thepMNTC.GFP expression cassette than in gerbil retinas carrying thepR2.1.GFP expression cassette (FIG. 4B). Likewise, GFP reporterexpression was detected sooner and in more cones in nonhuman primateretinas transduced with rAAV carrying the pMNTC.GFP expression cassetteas compared to NHP retinas transduced with the pR2.1 expression cassette(FIG. 5, n=4 eyes). In both gerbils and NHP, GFP was consistentlyobserved to be stronger from pMNTC than from pR2.1 throughout theduration of the study.

To determine the contribution of each of the elements in the pMNTCexpression cassette to the overall improvement in expression, a seriesof expression constructs were cloned in which each of the elements inpMNTC was substituted one-by-one with the corresponding element from thepR2.1 expression cassette. These constructs were then packaged intoAAV2-7m8 and delivered by intravitreal injection to the gerbil retina.Gerbil retinas were assessed 4 and 8 weeks later in vivo by in vivobioluminescence (IVIS imaging system, PerkinElmer), which provides aquantitative readout of reporter expression across the entire eye.

As expected, expression of the luciferase reporter under the control ofpMNTC was higher than expression of the luciferase reporter under thecontrol of pR2.1 (FIG. 6). Replacement of the pMNTC promoter sequencewith the pR2.1 promoter sequence having the most sequence homology to itreduced expression (construct pMNTC_pR2.1 L3′P), as did the inclusion ofpR2.1 promoter sequence that lies more distal to the 5′UTR of pR2.1(construct pMNTC_pR2.1-L5′P). Expression was also reduced by theintroduction into the pMNTC 5′UTR of two false start sequences (“AUG1”and “AUG2”) that were observed in the pR2.1 5′UTR (constructpMNTC_2.1-AUG1/2). Interestingly, expression was not reduced when thepMNTC 5′UTR was replaced with a modified pR2.1 5′UTR sequence in whichthese false starts had been removed (nucleotide 17 changed to C, nt 61and 62 changed to CA) (pMNTC_pR2.1-5′UTR), suggesting that the pR2.15′UTR would promote strong expression in cone cells but for the falseAUGs in the pR2.1 5′UTR element. Also interestingly, the pR2.1 intronappeared to provide more robust expression than the pSI chimeric intronof pMNTC, suggesting that inclusion of the pR2.1 intron in thepolynucleotide cassettes of the present disclosure may be used tofurther improve expression in cone cells. Lastly, removal of the L/Menhancer (found in both pR2.1 and pMNTC) reduced expression as well.While the polyA tailed seemed at first to also have a significant impacton expression, re-sequencing of the pMNTC construct comprising thispR2.1 element revealed that the polyA tail was not operably linked tothe transgene, thereby explaining why only background levels ofexpression were observed from this construct. Thus, the L/M opsin LCR,the inclusion of the M opsin core promoter rather than the L opsinpromoter, and the exclusion of false starts in the 5′UTR all contributeto the enhancement in gene expression achieved using the pMNTC promoter.

In conclusion, we have identified an AAV variant, the AAV variantcomprising a 7m8 peptide in the GH loop, which may be used for theintravitreal delivery of polynucleotides to retinal cones. Likewise, wehave identified a number of polynucleotide cassette elements that may beused to promote strong expression in cone photoreceptors. Together,these discoveries represent improvements that may facilitate thedevelopment of therapeutic agents for cone-associated disorders.

Materials and Methods

Transgene expression in vitro in WERI-RB-1 cells. WERI-Rb-1retinoblastoma cells expressing cone photoreceptor pigments cells aretransfected with a polynucleotide cassette of the present disclosureaccording to the method described by Shaaban and Deeb, 1998; IOVS39(6)885-896. The polynucleotide cassettes are transfected as plasmidDNA using well established techniques of molecular biology, such ascloning (Maniatis et al.) or via de novo DNA synthesis. All regulatoryelements are placed in the cassette and used to drive the enhanced GFPprotein. Plasmid DNA is then introduced into cells using establishedtechniques for non-viral transfection, for example using a lipid-basedtransfection reagent (Altogen Biosystems, NV) or Lipofectamine LTX (LifeTechnologies). Cells are then cultured for 72 hours and eGFP expressionis measured using flow cytometry and fluorescence microscopy. Transgeneexpression in cells transfected with the polynucleotide cassette of thepresent invention (i.e., constructs designed for cone photoreceptorexpression) is compared to the un-optimized counterparts (i.e., thosebased on pR2.1) and is found to be stronger from cassettes carryingimproved elements

In vitro expression is also evaluated using other mammalian cell linesthat express cone opsins, such as 661W cells (Tan et al., IOVS 2004;45(3) 764-768).

Similarly, in vitro expression is evaluated using non-photoreceptor celllines that have been engineered to express cone photoreceptor-specificproteins. Such a system has been described with HEK293 cells that havebeen genetically engineered to express CRX/Sp1 (Khani et al., IOVS 2007;48: 3954). Marker genes are also used (eGFP, dsRed, mCherry, luciferase)as well as physiologic genes (opsin, ACHR genes). Physiologic genes aretested by examining mRNA levels (e.g., by RT-PCR) or protein levels(e.g., by ELISA or Western blot).

Animal care. All experiments conformed to the principles regarding thecare and use of animals adopted by the American Physiological Societyand the Society for Neuroscience, and were approved by the InstitutionalAnimal Care and Use Committee (IACUC).

Small animal studies. The expression of the gene products encoded by thecoding sequence of the expression cassettes was evaluated in vivo inmice, rats, and gerbils. This was accomplished by intravitreal injectionin vivo of an rAAV preparation comprising the expression cassette (Li etal., 2008; Mol Vis 48: 332-338). Note that electroporation of plasmidDNA may be performed instead (Matsuda/Cepko).

Mouse studies. Mice used in this study were C57BL/6. Animals wereanesthetized with ketamine/xylazine (110 mg/kg intraperitoneal). Abeveled 34 gauge disposable needle loaded with test article was insertedinto the vitreous of the eye, and 5.04×1010 vector genomes of rAAV in avolume of 1.5μl was injected into the vitreous.

Gerbil and rat studies. Mongolian gerbils (Meriones unguiculatus) andbrown Norway rats were used in this study. Pupils were dilated with 10%phenylephrine and 0.5% tropicamide. Animals were anesthetized with anintraperitoneal or intramuscular injection of 0.1-0.2 mL of aketamine/xylazine solution (70 mg/mL ketamine and 10 mg/mL xylazine forrats; 25 mg/mL ketamine and 0.3 mg/mL xylazine for gerbils). A beveled34 gauge disposable needle loaded with test article in a 100 μL Hamiltonsyringe was inserted into the vitreous of the eye through the sclera atan optimized superior-temporal point about 1 mm from Limbus.1×1010-2×1010 vector genomes of test article (2×1010 vg of rAAV.GFP, or1.15×1010 vg of rAAV.luciferase) in a 5 uL volume was injected slowlywith a micro-injection pump into the vitreous, after which the needletip was held in the injected eye at the injected position for 10 secondsso as to ensure adequate test article dispensing. The needle was thenwithdrawn.

Non-human primate (NHP) studies. The polynucleotide cassettes andexpression vectors were also tested in large animals. This was done byusing AAV, for example using the techniques of Mancuso et al. Briefly,an AAV cassette was made, the AAV encapsidating the expression cassettewas manufactured, and the viral prep was injected intravitreally (up to170 uL in the vitreous) or subretinally (up to 3, 100 uL injections atdifferent locations; vitrectomy may be performed prior to injection) innonhuman primates. Expression was evaluated by reporter (GFP), colorERG, and/or behavioral testing using the Cambridge Color Test or onanimals trained to make a saccade (eye movement) when a target entersthe field of view. The saccades are monitored using an eye tracker.Prior to treatment animals are trained to perform a color vision test orto make a saccade when it sees a colored target. An ERG is performed toestimate the spectral sensitivity of the cones present. Data from thecolor vision test performance and the ERG provide evidence that theanimal is dichromatic (colorblind). For animals that receive a vectorcarrying the GFP gene, expression is monitored using fundus imaging withRetCam II or similar device under light that produces excitation of theGFP. For animals receiving a photopigment gene that differs in spectralsensitivity compared to the animal's endogenous pigments, expression ismonitored using the multifocal color ERG to measure spectral sensitivityat up to 106 different retinal locations, and by behavioral testing.

Baboons were sedated with 10-15 mg/kg ketamine following bysevofluorane. African Green monkeys were sedated with an intramuscularinjection of 5:1 ketamine:xylazine mix (0.2 ml/kg of 100 mg/ml ketamineand 20 mg/ml xylazine). Mydriasis was achieved with topical 10%phenylephrine. An eye speculum was placed in the eye to facilitateinjections. A drop of proparacaine hydrochloride 0.5% and then 5%betadine solution was applied, followed by a rinse with sterile saline.Baboons (FIG. 2) received 60 μl of a 3.4×10¹³ vg preparation of rAAV byintravitreal (ITV) injection to yield a final dose of 2.02×10¹² vg pereye. African Green monkeys received 50 uL of a 1×10¹³ preparation ofrAAV vector by ITV injection to yield a final dose of 5×10¹¹ vg per eye.ITV injections to the central vitreous were administered using a31-gauge 0.375 inch needle (Terumo) inserted inferotemporally at thelevel of the or a serrata ˜2.5 mm poster to the limbus under a surgicalmagnification to allow full visualization of extraocular and intraocularneedle placement. Central vitreous placement was confirmed by directobservation of the needle tip at the time of the injection. FollowingITV injections a topical triple antibiotic ointment was administered.

Slit-lamp biomicroscopy. The anterior segment of each monkey eye wasexamined by slit-lamp biomicroscopy during baseline screening and atweek 4 (day 28), week 8 (day 56) and week 12 (day 84) post-injection tomonitor inflammation. No abnormalities were observed.

NHP Necropsy and Eye Processing. Animals were euthanized withpentobarbital 12 weeks post intravitreal injection. Eyes were taggedwith a suture at the 12 o'clock position before enucleating and trimmingof extraocular tissues. Posterior cups were isolated by removing tissuesanterior to the limbus and fixed by immersion in 4% paraformaldehyde andstored in 70% ethanol.

Immunolabeling. Eyes were rehydrated into water then PBS buffer beforeflattening and delaminating retina as whole mounts. Preparations wereimaged by stereo fluorescence microscopy (Discovery FI V20, Carl ZeissMicroscopy, LLC, Thornwood, N.Y.) for GFP. Quadrant with fovea wasdetached from flat-mount, mounted under coverslip and imaged as fullmontage (5× tiling and stitching, Axio Observer Z1, Zeiss). Strip ofretina was isolated central at fovea out to periphery, cryoprotected insucrose and frozen in OCT. 8 μm sections were immunostained withantibodies to proteins enriched in specific retinal cell populations,including, L/M- and S-opsins, glutamine synthetase (GS), calbindin,rhodopsin (1D4), β-III Tubulin, Laminin, peanut agglutinin (PNA) and/orothers. GFP-tagged transgene containing cells were imaged bymultispectral analysis along with antibody probes and DIC (differentialinterference contract for topology) as flattened stacks of opticalplains through entire section (Axio Observer Z1, with Apotome, Zeiss).Cell analysis for transgene was optimized using morphology andcolocalization with probes.

Fundus examination and photography. Eye examination and fundusphotography of rat and gerbil retinas was performed using a PhoenixMicron IV fundus microscope. All animals received a baselinescreening/photographing to confirm ocular health, and then photographedat the designated timepoints to monitor the expression of the GFPtransgene. Any change to the optic nerves and retina or appearance ofgross lesions were recorded by a color fundus photography and expressionof GFP was visualized using fluorescence fundus imaging with afluorescein filter.

Retinal examination, fundus color and fluorescence photography, andautofluorescence OCT of NHP were performed by using a Topcon TRC-50EXretinal camera with Canon 6D digital imaging hardware and New VisionFundus Image Analysis System software and Spectralis OCT Plus. Allanimals received a baseline imaging. GFP expression was also documentedat week 2, 4, 8, and 12 post-intravitreal vector injection.

IVIS Imaging System. Expression of luciferase in the retina followingdelivery of rAAV.luciferase was quantified in vivo 2, 4 and 8 weekspost-intravitreal injection using an IVIS Imaging System. Gerbils wereinjected subcutaneously with 150 mg/kg luciferin (PerkinElmer) (15 mg/mlluciferin at a dose of 15 ml/kg). Approximately 22 minutes later,animals were sedated by inhalation of 4% isoflurane for 3-5 minutes.Immediately thereafter, animals were placed on the imaging platform inpairs, and the luminescence of the one eye of each animal quantifiedfollowed immediately by imaging of the contralateral eye. A naïve gerbilwas used as a negative standard, with background levels of luminescencetypically registering a luminescence of 1×10⁴ photons/second.Bioluminescence verification using a phantom mouse (XPM-2 Perkin Elmerphantom mouse for bioluminescence imaging) was performed prior toimaging to ensure calibration of the imaging system.

Immunohistochemistry. Mice were euthanized with a lethal dose of sodiumpentobarbital and tissues fixed via cardiac perfusion first with 0.13Mphosphate buffered saline (PBS) pH 7.2-7.4 containing 2 units of heparinper mL, followed by 4% paraformaldehyde (PFA) in PBS, followed by 4%paraformaldehyde plus 1% glutaraldehyde in PBS. Glutaraldehyde served tokeep the neural retina attached to the RPE so that the cone outersegments would remain intact. Each solution was warmed to ˜37° C. justprior to administration and ˜35-40 mL of perfusate was delivered at eachstage. Once the perfusion was stopped, the mouse was wrapped in a moistpaper towel and left to further fix for 2-3 hours before enucleation anddissection.

Permanent ink was used to mark the orientation of the eye, the anteriorsegment was removed, and the eye-cup was fixed in 4% PFA overnight at 4°C. and then stored in PBS at 4° C. Retinal whole-mounts were made byflattening the dissected retina between tissues soaked in 4% PFA for twohours and then transferring them to a culture plate for 6 more hours offixation. Afterward, the PFA was replaced with PBS containing 0.03%sodium azide (Sigma).

Antibody labeling was carried out on a rotating table shaker. To blocknon-specific labeling, whole mounts were incubated overnight at 4° C.with a solution containing 5% donkey serum (Jackson ImmunoResearch, Cat#004-000-120), 1 mg/ml BSA (Jackson ImmunoResearch, Cat #001-000-161),and 0.03% Triton X-100 in PBS (pH 7.4). The primary antibody used inthis study was rabbit anti red-green (L/M) opsin diluted 1:200(Millipore, Cat #AB5405. Specimens were washed in PBS 3 times for 30minutes each, then incubated at 4° C. overnight with DAPI(4′,6-diamidino-2-phenylindole, dihydrochloride 1:10,000; Invitrogen,Cat #D-21490) plus secondary antibodies. The secondary antibody for theL/M-opsin antibody was Alexa Fluor 488 labeled donkey anti-rabbitIgG(H+L) diluted 1:200 in antibody dilution buffer (Invitrogen, Cat#A21206). The incubation with secondary antibody was followed by three30 minute PBS washes, 30 minutes of post-fixation with 4%paraformaldehyde, and three more 30 minute PBS washes. Finally, theretinal slices were placed on slides with 2% DABCO in glycerol andcovered with cover slips.

Microscopy. Widefield images of mouse retina whole mounts were acquiredusing a Nikon Eclipse E1000 with a 20× (open-air) objective and cameraset with a 1.5× optical zoom. For each specimen, 50 optical sectionswere taken 0.5 μm apart and the M-opsin Z-stack was reconstructed inImageJ. The Z-stack was oriented so that the lengths of the outersegments were in plane, and the distance between where antibody stainingbegan and ended was measured as an estimate of the length of the outersegments. Further, a 3D projection of the Z-stack was generated and thenumber of cones with visible M-opsin in the outer segment could bequantified.

Confocal image slices were acquired using an Olympus FluoView™ FV1000.Sections were imaged using a 20× oil immersion lens (40 images taken 0.5μm apart) and the Z-stacks were reconstructed in ImageJ. Channelexposure levels were balanced within and across images using AdobePhotoshop. For the retinal whole mounts, images were taken using a 10×open-air lens and mosaics were constructed with Adobe Photoshop's nativemosaic construction software.

Experiments testing the tissue specificity of the polynucleotidecassettes. In this instance, a construct encoding GFP is injected viaone or more routes of administration, such as intravitreal, subretinal,or intravenously. The animal is then sacrificed and tissues are analyzedby qPCR—to detect DNA sequences indicating presence of the construct—andGFP expression—to detect areas where the construct is activelyexpressed. Whereas absence of DNA sequence indicates lack ofbiodistribution to a given tissue, the presence of DNA sequence togetherwith the lack of transgene expression (mRNA or protein level) indicatespresence of vector but lack of expression in that tissue. In this way,the level of specificity for cone photoreceptors can be established, andused to determine the utility of this invention in terms of restrictingexpression to target cone photoreceptor cells without expression innon-targeted tissues such as optic nerve, liver, spleen, or braintissue. Intravitreal AAV is known to biodistribute to the brain (Provostet al) so highly expressed, improved constructs for targeting conephotoreceptors would be useful to limit expression to target cells ofthe retina and limit potential adverse events associated with off-targettransgene expression.

The preceding merely illustrates the principles of the invention. Itwill be appreciated that those skilled in the art will be able to devisevarious arrangements which, although not explicitly described or shownherein, embody the principles of the invention and are included withinits spirit and scope. Furthermore, all examples and conditional languagerecited herein are principally intended to aid the reader inunderstanding the principles of the invention and the conceptscontributed by the inventors to furthering the art, and are to beconstrued as being without limitation to such specifically recitedexamples and conditions. Moreover, all statements herein recitingprinciples, aspects, and embodiments of the invention as well asspecific examples thereof, are intended to encompass both structural andfunctional equivalents thereof. Additionally, it is intended that suchequivalents include both currently known equivalents and equivalentsdeveloped in the future, i.e., any elements developed that perform thesame function, regardless of structure. The scope of the presentinvention, therefore, is not intended to be limited to the exemplaryembodiments shown and described herein. Rather, the scope and spirit ofthe present invention is embodied by the appended claims.

All publications and patent applications described herein are herebyincorporated by reference in their entireties.

1-29. (canceled)
 30. A method for delivering a polynucleotide of interest to a cone photoreceptor in a subject, the method comprising: delivering into the vitreous of the eye an effective amount of recombinant adeno-associated virus (rAAV) variant comprising the polynucleotide of interest, wherein: a) the rAAV variant comprises a variant AAV2 VP1 capsid protein comprising the amino acid sequence LGETTRP (SEQ ID NO: 11) inserted into the GH loop between amino acids 587 and 588 of the parental AAV2 VP1 capsid protein; and b) the therapeutic polynucleotide comprises a regulatory cassette operably linked to a polynucleotide encoding a therapeutic protein, wherein the regulatory cassette comprises a human L/M opsin Locus Control Region (“LCR”) enhancer and a truncated M-opsin promoter consisting of about 140 nucleotides upstream of the transcription start site.
 31. The method according to claim 30, wherein the variant AAV2 VP1 capsid protein comprises the amino acid sequence LALGETTRPA (SEQ ID NO: 13) inserted into the GH loop between amino acids 587 and 588 of the parental AAV2 VP1 capsid protein.
 32. The method according to claim 30, wherein the rAAV variant comprises a VP1 protein having a sequence identity of at least 80% to the polypeptide of SEQ ID NO:
 19. 33. The method according to claim 32, wherein the VP1 protein has a sequence identity of at least 95% to the polypeptide of SEQ ID NO:
 19. 34. The method according to claim 33, wherein the VP1 protein has a sequence identity of at least 99% to the polypeptide of SEQ ID NO:
 19. 35. The method according to claim 34, wherein the VP1 protein has a sequence identity of 100% to the polypeptide of SEQ ID NO:
 19. 36. The method according to claim 30, wherein the cone photoreceptor is a foveal cone.
 37. The method according to claim 30, wherein the subject is a primate.
 38. A method for expressing a gene product in a cone photoreceptor in a subject, the method comprising: delivering into the vitreous of the eye an effective amount of recombinant adeno-associated virus (rAAV) variant comprising a polynucleotide that encodes the gene product, wherein: a) the rAAV variant comprises a variant AAV2 VP1 capsid protein comprising the amino acid sequence LGETTRP (SEQ ID NO: 11) inserted into the GH loop between amino acids 587 and 588 of the parental AAV2 VP1 capsid protein; and b) the therapeutic polynucleotide comprises a regulatory cassette operably linked to a polynucleotide encoding a therapeutic protein, wherein the regulatory cassette comprises a human L/M opsin Locus Control Region (“LCR”) enhancer and a truncated M-opsin promoter consisting of about 140 nucleotides upstream of the transcription start site.
 39. The method according to claim 38, wherein the variant AAV2 VP1 capsid protein comprises the amino acid sequence LALGETTRPA (SEQ ID NO: 13) inserted into the GH loop between amino acids 587 and 588 of the parental AAV2 VP1 capsid protein.
 40. The method according to claim 38, wherein the rAAV variant comprises a VP1 protein having a sequence identity of at least 80% to the polypeptide of SEQ ID NO:
 19. 41. The method according to claim 40, wherein the VP1 protein has a sequence identity of at least 95% to the polypeptide of SEQ ID NO:
 19. 42. The method according to claim 41, wherein the VP1 protein has a sequence identity of at least 99% to the polypeptide of SEQ ID NO:
 19. 43. The method according to claim 42, wherein VP1 protein has a sequence identity of 100% to the polypeptide of SEQ ID NO:
 19. 44. The method according to claim 38, wherein the method further comprises detecting the expression of the polynucleotide in the cone photoreceptor.
 45. The method according to claim 38, wherein the cone photoreceptor is a foveal cone.
 46. The method according to claim 38, wherein the subject is a primate.
 47. A method for treating or preventing a cone-associated retinal disorder in a subject having or at risk for developing a cone-associated retinal disorder, the method comprising: administering intravitreally a recombinant adeno-associated virus (rAAV) variant comprising a therapeutic polynucleotide in an amount effective to treat or prevent the cone-associated retinal disorder, wherein: a) the rAAV variant comprises a variant AAV2 VP1 capsid protein comprising the amino acid sequence LGETTRP (SEQ ID NO: 11) inserted into the GH loop between amino acids 587 and 588 of the parental AAV2 VP1 capsid protein; and b) the therapeutic polynucleotide comprises a regulatory cassette operably linked to a polynucleotide encoding a therapeutic protein, wherein the regulatory cassette comprises a human L/M opsin Locus Control Region (“LCR”) enhancer and a truncated M-opsin promoter consisting of about 140 nucleotides upstream of the transcription start site.
 48. The method according to claim 47, wherein the variant AAV2 VP1 capsid protein comprises the amino acid sequence LALGETTRPA (SEQ ID NO: 13) inserted into the GH loop between amino acids 587 and 588 of the parental AAV2 VP1 capsid protein.
 49. The method according to claim 47, wherein the VP1 protein has a sequence identity of at least 80% to the polypeptide of SEQ ID NO:
 19. 50. The method according to claim 49, wherein the VP1 protein has a sequence identity of at least 95% to the polypeptide of SEQ ID NO:
 19. 51. The method according to claim 50, wherein the VP1 protein has a sequence identity of at least 99% to the polypeptide of SEQ ID NO:
 19. 52. The method according to claim 51, wherein the VP1 protein has a sequence identity of 100% to the polypeptide of SEQ ID NO:
 19. 53. The method according to claim 47, wherein the retinal disorder is a cone-associated disorder.
 54. The method according to claim 53, wherein the cone-associated disorder is selected from the group consisting of rod-cone dystrophy; cone-rod dystrophy; progressive cone dystrophy; retinitis pigmentosa (RP); Stargardt Disease; macular telangiectasia, Leber hereditary optic neuropathy, Best's disease; adult vitelliform macular dystrophy; X-linked retinoschisis; a color vision disorder; age-related macular degeneration; wet age-related macular degeneration; geographic atrophy; diabetic retinopathy; a retinal vein occlusion; retinal ischemia; Familial Exudative Vitreoretinopathy (FEVR); COATs disease; and Sorsby's fundus dystrophy.
 55. The method according to claim 47, wherein the method further comprises identifying the subject as having a cone-associated disorder.
 56. The method according to claim 47, wherein the method further comprises detecting an improvement in vision following the administering step.
 57. The method according to claim 47, wherein the subject is a primate.
 58. The method according to claim 47, wherein the retinal disorder is a color vision disorder.
 59. The method according to claim 58, wherein the color vision disorder is blue cone monochromacy.
 60. The method according to claim 58, wherein the color vision disorder is color vision deficiency. 